Full text of DOI:10.1359/jbmr.2001.16.2.299

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 2, 2001
© 2001 American Society for Bone and Mineral Research
Strontium Ranelate Increases Cartilage Matrix Formation
Y. HENROTIN,1 A. LABASSE,1 S.X. ZHENG,1 Ph. GALAIS,2 Y. TSOUDEROS,2 J.M. CRIELAARD,1 and
J.Y. REGINSTER1
ABSTRACT
Based on previous studies showing that strontium ranelate (S12911) modulates bone loss in osteoporosis, it
could be hypothesized that this drug also is effective on cartilage degradation in osteoarthritis (OA). This was
investigated in vitro on normal and OA human chondrocytes treated or not treated with interleukin-1␤
(IL-1␤). This model mimics, in vitro, the imbalance between chondroformation and chondroresorption
processes observed in vivo in OA cartilage. Chondrocytes were isolated from cartilage by enzymatic digestion
and cultured for 24–72 h with 10؊4–10؊3 M strontium ranelate, 10؊3 M calcium ranelate, or 2 ⅐ 10؊3 M SrCl2
with or without IL-1␤ or insulin-like growth factor I (IGF-I). Stromelysin activity and stromelysin quanti-
tation were assayed by spectrofluorometry and enzyme amplified sensitivity immunoassay (EASIA), respec-
tively. Proteoglycans (PG) were quantified using a radioimmunoassay. Newly synthesized glycosaminoglycans
(GAGs) were quantified by labeled sulfate (Na235SO4) incorporation. This method allowed the PG size after
exclusion chromatography to be determined. Strontium ranelate, calcium ranelate, and SrCl2 did not modify
stromelysin synthesis even in the presence of IL-1␤. Calcium ranelate induced stromelysin activation whereas
strontium compounds were ineffective. Strontium ranelate and SrCl2 both strongly stimulated PG production
suggesting an ionic effect of strontium independent of the organic moiety. Moreover, 10؊3 M strontium
ranelate increased the stimulatory effect of IGF-I (10؊9 M) on PG synthesis but did not reverse the inhibitory
effect of IL-1␤. Strontium ranelate strongly stimulates human cartilage matrix formation in vitro by a direct
ionic effect without stimulating the chondroresorption processes. This finding provides a preclinical basis for
in vivo testing of strontium ranelate in OA. (J Bone Miner Res 2001;16:299–308)
Key words: strontium ranelate, chondrocytes, proteoglycans, stromelysin, osteoarthritis
tant pathophysiological process. The levels and activity of
stromelysin were found to be high in OA cartilage, synovia,
and synovial fluid,(5–7) whereas PG synthesis and content
decreased with lesion severity.(8) The metabolic disruption
is characterized by PG release, fixed charge density, and
cation concentration decrease.(9) As the rate of PG synthesis
decreases when the ionic strength is below the physiological
strength, cation administration could have beneficial effects
INTRODUCTION
ARTILAGE IS constantly being turned over in the balance
between extracellular matrix synthesis and degradation.
Chondroformation is stimulated by growth factors such as
insulin-like growth factor I (IGF-I).(1) Chondroresorption,
which involves metalloproteinases like stromelysin,(2,3) is
stimulated by cytokines such as interleukin-1␤ (IL-1␤).(4) In
C
osteoarthritis (OA), this delicate equilibrium is progres- on OA chondrocyte metabolism.(10,11) Strontium, a divalent
sively disrupted, leading to an excess of tissue degradation cation, has shown evidence of beneficial effects on bone
and, finally, tissue disappearance. The imbalance between metabolism(12) and interference in cartilage metabolism.(13)
stromelysin and proteoglycans (PGs) constitutes an impor- Strontium ranelate (S12911), an uncoupling agent devel-
1Bone and Cartilage Metabolism Research Unit, University Hospital, Lie`ge, Belgium.
2Servier, Courbevoie, France.
299

300
HENROTIN ET AL.
oped as a preventive and curative treatment of postmeno-
pausal osteoporosis,(12,14–16) prevents bone loss induced by
estrogen deficiency by inhibiting bone resorption and stim-
ulating bone formation.(17)
The present study investigated the effects of strontium
ranelate on stromelysin and PG synthesis in human chon-
drocytes cultured with or without IL-1␤ or IGF-I. In chon-
drocyte culture, IL-1␤ reproduces the decoupling effect
leading to a decrease in PG and an increase in stromelysin
synthesis,(4,18) whereas IGF-I stimulates PG production.(1)
The effects of strontium ranelate were compared with the
effects of calcium ranelate and strontium chloride (SrCl2),
which were used as controls.
FIG. 1. Structure of strontium ranelate and calcium ranelate.
MATERIALS AND METHODS
gestion of the tissue were 95% viable (trypan blue test). CPs
were extracted before PG and DNA analysis (CP extract).
They were washed twice with Hanks’ buffered saline solu-
tion (HBSS; Gibco) and homogenized by ultrasonic disso-
ciation (10 s, 50 W/cm2) in phosphate-buffered saline, pH
7.5, containing proteinase inhibitors. All the chemicals for
which the source was not mentioned were of the purest
grade commercially available.
Human chondrocyte culture
Freshly isolated human cartilage chondrocytes were cul-
tured at a high density for a short-term period (24–72 h) to
retain their phenotype. Cartilage samples were obtained
from the knees of 13 cadavers (23–83 years old) immedi-
ately after death. They were excised from the superficial and
medium layer of cartilage, avoiding the calcified layer.
Pathological cartilage modifications were evaluated on fem-
oral and patellar articular surfaces using the Moskowitz
scale.(8) Each culture was run with chondrocytes from a
single patient. The cartilage was cut into small fragments.
Chondrocytes were isolated from their extracellular matrix
by enzymatic digestion. Cartilage fragments (3 g) were first
treated (30 minutes, 37°C, 200 rpm) with 10 ml hyaluron-
idase solution (Sigma Chemie, Bornem, Belgium) previ-
ously dissolved (0.5 mg/ml) in Dulbecco’s modified Eagle’s
medium (DMEM; Biowhittaker, Brussels, Belgium). Carti-
lage fragments were then treated (1 h at 37°C) with 10 ml
pronase solution (1 mg/ml in DMEM; Merck-Belgolabo,
Overijse, Belgium). Finally, they were incubated (20 h,
37°C, 200 rpm) with 10 ml collagenase (Sigma Chemie)
solution (1 mg/ml in DMEM containing 1% Ultroser G, a
serum substitute supplied by Gibco, Gent, Belgium). The
cells were filtered through a nylon mesh (70 ␮m), washed
three times, counted (range, 1–2 ⅐ 106 cells/ml), and resus-
pended in 1 ml of adequate culture medium (CM; DMEM
supplemented with 1% insulin, transferin, and seleium
(ITSϩ) or TSϩ, 2 mM glutamine, and 50 ␮g/ml ascorbate).
ITSϩ is a culture supplement containing 0.625 mg/ml in-
sulin, 0.625 mg/ml transferrin, 0.625 ␮g/ml selenious acid,
125 mg/ml bovine serum albumin (BSA), and 0.530 mg/ml
linoleic acid. When chondrocytes were incubated with
IGF-I, ITSϩ was replaced by TSϩ (TSϩ is ITSϩ without
insulin). DMEM contained 1.8 mM calcium. Cells were
kept in this CM for 48 h to eliminate in vivo contamination
Culture treatment
Human chondrocytes (1–2 ⅐ 106 cells/ml) were cultured
for 24–72 h in the absence or presence of strontium ranelate
(15 ⅐ 10Ϫ4–10Ϫ3 M), SrCl2 (2 ⅐ 10Ϫ3 M), or calcium rane-
late (10Ϫ3 M) with or without IL-1␤ (10Ϫ11 M and 10Ϫ10
M) or IGF-I (10Ϫ3 M and 10Ϫ8 M). The tested compounds
were directly dissolved in the CM, which was then sterilized
by filtration before cellular culture. Strontium ranelate
(S12911; Servier, Courbevoie, France) is made up of two
stable strontium atoms and ranelic acid as carrier (5-
{bis(carboxymethyl)amino}-2-carboxy-4-cyano-3-thio-
phenacetic acid). In calcium ranelate (S12911–0), strontium
atoms are replaced by two calcium atoms (Fig. 1). Three
flasks were used for each concentration of the drug and for
the corresponding controls.
DNA assay
Chondrocyte DNA content is correlated directly to the
cell number of each culture. DNA content was measured in
the CP extract using a fluorometric method.(19) This mea-
surement ensures elimination of result variations caused by
the different number of chondrocytes in each culture.
Stromelysin activity measurement
Stromelysin activity released in the CM was measured on
with drugs that the donors may have taken before death. resorufin-labeled casein from cow’s milk (Boehringer,
After this washout period, cells were seeded in 10 ml Mannheim, Germany) using a spectrofluorometric method.
polypropylene Falcon tubes (1–2 ⅐ 106 cells/ml) and main- Latent stromelysin (prostromelysin) was activated or not
tained under constant agitation (100 rpm) in a 95% air/5% activated by p-aminophenylmercuric acetate(20) (APMA;
CO2 atmosphere. At the end of each culture period (1, 2, or Sigma Chemie) treatment of the CM (0.5 mM for 4 h at
3 days), cells (chondrocyte pellet [CP]) and supernatant 37°C). APMA-activated CM (100 ␮l) was then incubated
(CM) were separated by centrifugation (1000 rpm for 5 with 20 ␮g resorufin-labeled casein for 18 h at 37°C in a
minutes). The chondrocytes recovered after collagenase di- standard buffer (Tris-HCl 0.2 M, pH 7.5) containing 0.02 M

STRONTIUM RANELATE INCREASES CHONDROFORMATION
301
CaCl2. The enzymatic reaction was stopped by adding tri- trypsin inhibitor, 0.01 M EDTA, and 6.7 ⅐ 10Ϫ3 M sodium
chloroacetic acid at a final concentration of 1.8% and 20 azide. CP previously washed three times with HBSS was
mM EDTA. The samples were centrifuged at 7000g for 15 extracted (24 h, at 4°C, 100 rpm) by 500 ␮l of guanidine
minutes. Cleaved casein was then measured fluorometri- hydrochloride (GuHCl, 4 M, in 0.5 M sodium acetate solu-
cally in 600 ␮l of supernatant previously buffered with 900 tion containing protease inhibitors, pH 5.8). Three CP ex-
␮l of Tris-HCl (0.5 M, pH 8.8). Excitation and emission tracts treated in the same conditions were pooled and dia-
wavelengths were 574 nm and 584 nm, respectively. Paral- lyzed against distilled water and Tris-HCl, 0.05 M (pH 9).
lel samples containing 20 mM EDTA added before incuba-
Radioactivity was measured in the samples using an LKB
tion were measured similarly, providing blank values. A 1214 RackBeta liquid scintillation counter (Amersham In-
standard curve obtained by incubation of increased quanti- ternational, Brussels, Belgium).
ties of purified stromelysin (Biosource Europe, Nivelle,
Belgium) allowed the conversion of the measured fluores-
cence values into the stromelysin concentration equivalent
Determination of the PG size
(␮g Eq strom/ml). The molecular weight of the detected
The size of the PG produced by the chondrocytes was
proteinase was assessed by electrophoresis on a casein-
determined by chromatography. CM and CP extract were
eluted on gel filtration chromatography under associative
(21)
containing gel (Zymogram) as previously described.
conditions. These conditions protect PG complexes from
Stromelysin quantitation
hydrolysis and allow PG separation according to their mo-
lecular weight. For analytical purposes, a column (0.8 cm ϫ
60 cm) was packed with Sepharose CL2B (Pharmacia,
Uppsala, Sweden). The column was equilibrated and eluted
with 0.05 M Tris-HCl (pH 9) containing enzymatic inhibi-
tors. The flow rate was 6 ml/h and 1-ml fractions were
collected. The column was calibrated under the same con-
ditions with a 1-ml sample adjusted to 20,000 cpm
(Na235SO4). The chromatogram was established by deter-
mining the radioactivity in 0.5 ml of each fraction. The
chromatographic profile of the 35S-GAG was characterized
by a distribution coefficient [Kd ϭ (Ve-V0)/(Vs-V0)], where
V0 is the void volume of the column, Vs the total volume of
the column, and Ve is the elution volume of the molecule
under consideration. The peak fraction containing 35S-PG
aggregates and Na235SO4 were used to determine V0 and VS,
respectively. Radioactivity allowed repartition of PG into
three groups: PG-hyaluronic acid complexes (Kd Յ 0.13),
PG monomers and small complexes (0.13 Ͻ Kd Ͻ 0.7), and
small PG and PG fragments (Kd Ն 0.7).
Total stromelysin released in the CM was assayed using
an enzyme amplified sensitivity immunoassay (EASIA;
Biosource Europe). In this immunoassay, monoclonal anti-
bodies are directed against prostromelysin, activated
stromelysin, and stromelysin bound to tissue inhibitor of
metalloproteases (TIMP)-1 and TIMP-2. Total stromelysin
corresponds to the level of stromelysin synthesized in the
CM including prostromelysin, activated stromelysin, and
stromelysin bound to TIMP-1 and TIMP-2. The method was
linear between 5 and 20 ng/ml with a limit of detection of
2.5 ng/ml.
PG quantitation
PGs were quantified in CM and CP extracts by radioim-
munoassay (RIA) as previously described.(22,23) Rabbit
polyclonal antibodies used are specific to the antigenic
determinant of the PG core protein. No cross-reaction was
observed with glycosaminoglycans (GAGs) such as chon-
droitin sulfate or keratan sulfate or with PG treated with
various enzymes. Only treatment with proteolytic enzymes
(papain, trypsin, and chymotrypsin) abolished or diminished
immunoreactivity as opposed to treatment with chondroiti-
Expression of the results and statistical analysis
The results were expressed as the rate of stromelysin or
nase ABC and neuraminidase. Antiserum did not cross- PG released into the CM or present inside the CP per
react with other cartilage matrix constituents such as type II, microgram of DNA. The values presented are the cumula-
type IX, and type XI collagens. The lack of cross-reaction tive amounts of stromelysin or PG found in the CM at the
with bone PG or skin, cornea, liver, heart, and lung tissue end of the specified culture period. Total PG production
extracts, showed that the antiserum used in the study con- (TPG) was calculated by adding the results measured for the
tains only specific chondrocyte-aggrecan antibodies.
CM and the corresponding CP. Cumulative release rates of
35S-GAG were obtained by adding the amounts found in the
CM at the end of each incubation period. The mean Ϯ SD
of each variable was calculated. Comparison of mean values
Sulfated GAG quantitation
PG monomers are made up of sulfated GAG bound on a was performed for each culture individually using the un-
linear protein core. The quantitation of the newly synthe- paired Student’s t-test. Production curves were compared
sized PG was carried out by determination of radioactivity for each experimental condition by means of Zerbe’s ran-
incorporated in sulfated GAG (35S-GAG). Na235SO4 was domization test.(24) Values were treated with a generalized
added to the CM (5 ␮Ci/ml) during the last 24 h of the linear mixed model to compare the effects of strontium
culture. To eliminate unlabeled Na235SO4, CM was dialyzed ranelate, calcium ranelate, and strontium chloride on young
three times against Tris-HCl 0.05 M (pH 9) containing normal and old OA cartilage groups; treated groups were
protease inhibitors. The protease inhibitors used throughout compared between themselves and with the controls. Cor-
the study were 5 ⅐ 10Ϫ8 M 6-aminohexanoic acid, 5 ⅐ 10Ϫ8 M relations between the variables were investigated by a linear

302
HENROTIN ET AL.
TABLE 1. DNA CONTENTS
Controls
S12911
S12911-0
10Ϫ3
SrCl2
T0h
T72h
10Ϫ4
M
5 ⅐ 10Ϫ4
M
10Ϫ3
M
M
2 ⅐ 10Ϫ3
M
Without IL-1␤
With IL-1␤
12.5 Ϯ 1.3
12.9 Ϯ 2.0
13.0 Ϯ 0.8
13.1 Ϯ 1.0
12.9 Ϯ 1.1
12.7 Ϯ 1.3
12.3 Ϯ 0.3
13.9 Ϯ 0.3
12.1 Ϯ 0.7
13.4 Ϯ 0.4
12.3 Ϯ 0.2
11.5 Ϯ 0.7
11.1 Ϯ 0.7
13.0 Ϯ 1.2
Effects of strontium ranelate (S 12911), calcium ranelate (S 12911-0) or SrCl2 on DNA (␮g) contained in CP after 72h of normal human
chondrocyte culture. The DNA content at the begining (T0h) and the end of the culture (T72h) were also compared for the controls. Results
are mean values and SD of triplicate primary chondrocyte cultures in a representative experiment.
FIG. 2. Effect of increased amounts of strontium ranelate, SrCl2, or calcium ranelate on stromelysin activity and synthesis by normal human
chondrocytes. Primary chondrocytes were cultured for 72 h in the absence (-E-) or presence of 10Ϫ4 M (-ϫ-), 5 ⅐ 10Ϫ4 M (-ࡗ-) or 10Ϫ3 M (-Ⅺ-)
strontium ranelate, 2 ⅐ 10Ϫ3 M SrCl2 (-F-), or 10Ϫ3 M calcium ranelate (-ƒ-). Results are the mean values of triplicate primary chondrocyte
cultures in a representative experiment and error bars represent SD. The values represent the cumulative amounts of stromelysin over the specified
culture periods. Differences between curves are tested by the Zerbe test.
regression analysis and a one-way analysis of variance
(ANOVA) was performed.
Stromelysin synthesis and activity
In basal conditions (no IL-1␤ stimulation), stromelysin
activity measured in the CM of unactivated chondrocytes
was very low (Fig. 2) or undetectable (Fig. 3), depending on
the donor. However, stromelysin synthesis increased lin-
early with culture duration. Strontium ranelate and SrCl2 did
not activate stromelysin, whereas calcium ranelate induced
enzyme activation in the culture where stromelysin activity
was detectable (p Ͻ 0.001; Fig. 2).
RESULTS
Chondrocyte viability
Preliminary tests (trypan blue, 51Cr release) showed the
absence of cytotoxicity of the tested compounds. Cell via-
bility was always superior to 90% whatever the culture
treatment. The DNA content remained stable during the
APMA-activated stromelysin activity measured in the
CM increased as a function of culture duration but was not
culture period (no significant difference between 0 and 72 h) affected by the presence of calcium or strontium compounds
and was not significantly affected by the presence of stron- (Fig. 2). Furthermore, strontium ranelate, calcium ranelate,
tium ranelate (10Ϫ4–10Ϫ3 M), calcium ranelate (10Ϫ3 M), or and SrCl2 did not increase stromelysin protein synthesis
SrCl2 (2 ⅐ 10Ϫ3 M; Table 1).
(Fig. 2).

STRONTIUM RANELATE INCREASES CHONDROFORMATION
303
FIG. 3. Stromelysin activity
and synthesis in the CM by un-
stimulated or IL-1␤–stimulated
(10Ϫ11 M) normal human chon-
drocytes were measured after
72 h of treatment with 10Ϫ3
M
strontium ranelate (S12911), 2 ⅐
10Ϫ3 M SrCl2, and 10Ϫ3 M cal-
cium ranelate (S12911–0). A sig-
nificant increase in the stromely-
sin activity was observed in the
presence of 10Ϫ3
M calcium
ranelate in comparison with un-
activated stromelysin. Response
is significant with p Ͻ 0.05.
IL-1␤ stimulated stromelysin synthesis and activity mixed model; Table 2). Strontium ranelate (10Ϫ4–10Ϫ3 M)
with a maximal effect after 72 h of incubation (p Ͻ 0.01; significantly increased TPG (0.0005 Ͻ p Ͻ 0.0001) in
Fig. 3). The 72-h incubation period was retained to test normal chondrocyte cultures. No significant increase was
the compounds with IL-1␤ stimulation. Strontium com- observed at the concentration of 10Ϫ4 M when strontium
pounds did not significantly modify IL-1␤–stimulated ranelate was tested on OA cartilage, whereas higher con-
stromelysin synthesis and activity whereas calcium rane- centrations (5 ⅐ 10Ϫ4 M and 10Ϫ3 M) induced a significant
late increased IL-1␤–induced stromelysin activity (p Ͻ increase in TPG (0.002 Ͻ p Ͻ 0.02). SrCl2 (2 ⅐ 10Ϫ3 M) is
0.05; Fig. 3).
as efficient as strontium ranelate in increasing TPG pro-
duced by normal and OA chondrocytes (Table 2). Never-
theless, strontium ranelate increased PG contained in the CP
of normal chondrocyte cultures whereas 2 ⅐ 10Ϫ3 M SrCl2
did not significantly modify this parameter. Calcium rane-
late (10Ϫ3 M) also increased TPG in normal chondrocyte
cultures to the same extent as strontium ranelate (Table 2).
PG production
In control conditions and in strontium ranelate–treated
cultures, the PGs that were released in the CM (Fig. 4A) and
contained in the CP (Fig. 4B) of normal chondrocyte cul-
tures increased as a function of the culture duration. After
24 h of culture, strontium ranelate did not significantly
modify PG production by normal chondrocytes whatever
the concentration used. After 48 h of culture, strontium
ranelate significantly increased the amount of PG released
in the CM only at the concentration of 10Ϫ3 M and the PG
contained in the CP at the concentrations of 5 ⅐ 10Ϫ4 M and
10Ϫ3 M. After 72 h of culture, strontium ranelate dose-
dependently increased the PG released in the CM (r ϭ 0.92;
p Ͻ 0.01). This increase was significant at concentrations of
5 ⅐ 10Ϫ4 M and 10Ϫ3 M in the CP (p Ͻ 0.05). TPG
production was strongly and significantly increased (ap-
proximately 20–35%) by 5 ⅐ 10Ϫ4 M or 10Ϫ3 M strontium
ranelate after 48 h and 72 h of culture (data not shown).
The effects of strontium ranelate were then compared
between normal and OA chondrocyte cultures. Six different
cartilage samples were used. They were excised from 3
normal young donors (Ͻ40 years old) and 3 OA donors
(Ͼ60 years old). OA samples showed one or more lesions
that scored III on the Moskowitz scale.(8) As expected,
chondrocytes isolated from the cartilage of normal donors
always produced higher amounts of PG than OA chondro-
Chondrocyte responses to IGF-I and IL-1␤
The effects of strontium ranelate on chondrocyte re-
sponses to IGF-I and IL-1␤ were studied in normal human
chondrocyte cultures. As expected, IGF-I dose-dependently
stimulated TPG (p ϭ 0.02) whereas IL-1␤ drastically in-
hibited PG synthesis after 72 h of culture. IGF-I (10Ϫ9
M
and 10Ϫ8 M) increased TPG by 15% and 40%, respectively.
When IGF-I (10Ϫ9 M) and strontium ranelate (5 ⅐ 10Ϫ4 M or
10Ϫ3 M) were added simultaneously, the stimulatory effect
of IGF-I was higher than the effect of IGF-I alone (0.03 Ͻ
p Ͻ 0.004; unpaired Student’s t-test; Fig. 5). No similar
effects were observed in the presence of 10Ϫ8 M IGF-I.
In the presence of 10Ϫ11 M or 10Ϫ10 M IL-1␤, TPG
drastically decreased by 60% and 68%, respectively. As
shown in Fig. 6, 10Ϫ3 M strontium ranelate did not signif-
icantly modify the IL-1␤–inhibiting effect on TPG.
PG size
In the culture conditions used, the newly synthesized PGs
cytes (Table 2). The PG amount released into the CM and were built up with 35S-GAG. The amounts of 35S-GAG
contained in the CP after 72 h of culture with or without incorporated into the newly synthesized PGs during the last
strontium ranelate was significantly lower in the OA group 24 h of 1, 2, or 3 days of normal human chondrocyte
than in the normal group (p Ͻ 0.001; generalized linear cultures are presented in Fig. 7.

304
HENROTIN ET AL.
35S-GAG eluted with a Kd Յ 0.13 increased, with a Kd
between 0.13 and 0.7 it was not modified, and with a Kd Ն
0.7 it decreased (Table 3, Fig. 8A).
PG contained in the cellular phase: The amount of 35S-
GAG contained in the CP of the culture increased as a
function of the culture duration (Fig. 7B). Strontium rane-
late (10Ϫ3 M) and SrCl2 (2 ⅐ 10Ϫ3 M) significantly increased
the amount of 35S-GAG contained in the CP.
The chromatographic profile of 35S-GAG contained in the
CP of untreated chondrocyte cultures presented two major
peaks (Fig. 8B). In the untreated cultures, 29–37% of the
35S-GAG were eluted with a Kd Յ 0.13, the majority of the
35S-GAG (43–52%) was eluted with a Kd between 0.13 and
0.7, whereas only 11–28% was eluted with a Kd Ն 0.7
(Table 3). As in the CM, the high-molecular weight PG
complex rate contained in the CP increased with the culture
duration whereas the rate of small PG and PG fragments
decreased. Strontium ranelate (10Ϫ3 M) and SrCl2 (2 ⅐ 10Ϫ3
M) treatments increased the PG contained in the CP without
any effect on the PG size (Table 3; Fig. 8B).
DISCUSSION
In basal conditions and without any treatment, stromely-
sin activity was very low whereas stromelysin production
increased linearly with the culture duration. This finding
suggests that stromelysin is produced as prostromelysin
(inactive form) and that only a small part of the proenzyme
is activated. Interindividual variability in stromelysin syn-
thesis and activity was observed (Figs. 2 and 3 after 72 h).
Stimulation of the chondrocytes by IL-1␤ drastically in-
creased stromelysin activity released in the CM. The mech-
anism involved may be the plasminogen activator/plasmin
system, which can transform the proenzyme to the active
form of stromelysin.(25) This hypothesis agrees with previ-
ous studies in which IL-1␤ was shown to increase plasmin-
ogen activator synthesis by the main cells of the joint,
FIG. 4. Effect of strontium ranelate on human articular chondrocyte
cultures. Normal human chondrocytes were incubated for the time
specified in the absence (control condition) or presence of varying
concentrations of strontium ranelate. At the end of the culture period,
CM and CP were separated by centrifugation and were assayed for PG
by a specific RIA. The data are expressed as amounts of PG found in
the CM or in the CP per micrograms of DNA and presented as the mean
and SD of triplicate cultures. Statistical significance in comparison to
the controls, *p Ͻ 0.05.
(26,27)
including human chondrocytes.
Strontium ranelate and SrCl2 did not activate stromelysin
whereas calcium ranelate increased enzyme activity only in
basal conditions. Calcium ranelate must act on the proen-
zyme activation process, because it did not increase strome-
lysin synthesis and did not modify the activity of APMA-
PG released in the CM: In the absence of treatment, the activated stromelysin. This hypothesis agrees with a
daily production of 35S-GAG released in the CM reached a previous study that reported that Ca2ϩ is required for
maximum after 48 h of culture (Fig. 7A, same slope after prostromelysin activation and stability.(20)
24 h and after 48 h). Strontium ranelate (10Ϫ3 M) and SrCl2
In the condition used, chondrocytes cultured without any
(2 ⅐ 10Ϫ3 M) significantly increased the 35S-GAG synthesis treatment produced spontaneously aggregating PG and type
after 48 h and 72 h of incubation. II collagen. The TPG measured in the cultures from the
The newly synthesized 35S-GAG released in the control different chondrocyte donors shows interindividual varia-
CM were eluted through a CL2B chromatographic column tion (Table 2; Figs. 4–6; after 72 h without any treatment).
in two major peaks (Fig. 8A). At each culture period, a large A large part of the newly synthesized PG (10–20%) was
part of the newly synthesized 35S-GAG was eluted with a Kd contained in the CP of the culture, suggesting the formation
Յ 0.13 (Table 3). The percentage of 35S-GAG eluted with of a new matrix surrounding the chondrocytes. The quali-
a Kd Յ 0.13 (high-molecular weight PG complexes) in- tative analysis of the newly synthesized PG showed that the
creased with the culture duration (36–49%) whereas the majority of the PG released in the CM (56%) and extracted
35S-GAG eluted with a Kd Ն 0.7 (small PG and PG frag- from the CP (57%) is high-molecular weight PG complexes.
ments) decreased (37–25%). In the presence of 10Ϫ3
M
Strontium ranelate (10Ϫ3 M) strongly stimulated the syn-
strontium ranelate or 2 ⅐ 10Ϫ3 M SrCl2, the percentage of the thesis of PG by human chondrocytes in vitro. This cartilage

STRONTIUM RANELATE INCREASES CHONDROFORMATION
305
TABLE 2. PG PRODUCTION MEASURED BY RIA
S12911
S12911-0
10Ϫ3
SrCl2
Controls
10Ϫ4M
5 ⅐ 10Ϫ4
M
10Ϫ3
M
M
2 ⅐ 10Ϫ3
M
Proteoglycans contained in the culture supernatants
Normal
OA
622 Ϯ 184
212 Ϯ 68
707 ؎ 146
p ϭ 0.0012
216 Ϯ 62
814 ؎ 191
p ϭ 0.0001
267 ؎ 91
800 ؎ 176
p ϭ 0.0001
292 ؎ 90
752 ؎ 145
p ϭ 0.0001
—
844 ؎ 206
p ϭ 0.0001
302 Ϯ 12
p ϭ 0.86
p ϭ 0.03
p ϭ 0.0088
p ϭ 0.0006
Proteoglycans contained in the cellular phases
Normal
OA
68 Ϯ 25
36 Ϯ 15
73 Ϯ 24
p ϭ 0.13
36 Ϯ 15
82 ؎ 20
p ϭ 0.0003
41 Ϯ 17
85 ؎ 18
p ϭ 0.0001
45 Ϯ 18
82 ؎ 26
p ϭ 0.0001
—
71 Ϯ 20
p ϭ 0.3
40 Ϯ 12
p ϭ 0.9
p ϭ 0.13
p ϭ 0.27
p ϭ 0.24
Total proteoglycan production
Normal
OA
686 Ϯ 210
248 Ϯ 81
780 ؎ 167
p ϭ 0.0005
253 Ϯ 75
894 ؎ 210
p ϭ 0.0001
309 ؎ 105
885 ؎ 194
p ϭ 0.0001
362 ؎ 156
833 ؎ 169
p ϭ 0.0001
—
914 ؎ 223
p ϭ 0.0001
340 ؎ 115
p ϭ 0.85
p ϭ 0.02
p ϭ 0.002
p ϭ 0.0007
Effects of strontium ranelate (S 12911), calcium ranelate (S 12911-0), or SrCl2 on PG production (ng/␮g DNA) by normal or OA
human chondrocytes at 72h. Values are the means and SD of three separate chondrocyte cultures. Values with p Ͻ 0.05 are significantly
different from the corresponding controls (in bold).
FIG. 6. Effects of strontium ranelate on normal human chondrocyte
FIG. 5. Effect of strontium ranelate on normal human chondrocyte
response to IGF-I. At the concentrations of 10Ϫ9 M and 10Ϫ8 M, IGF-I
response to IL-1␤: TPG produced by human chondrocytes cultured for
72 h in the absence (black column) or in the presence of 10Ϫ3
M
significantly stimulated TPG (sum of the PG amount released in the
CM and contained in the corresponding CP) of normal human chon-
drocytes cultured for 72 h in the absence (black column) or in the
strontium ranelate (gray column) or 2 ⅐ 10Ϫ3 M SrCl2 (white column).
presence of 10Ϫ3 M strontium ranelate (gray column) or 2 ⅐ 10Ϫ3
M
Svensson(29) who showed that SrCl2 inhibited the synthesis
of GAG and collagen in freshly isolated rat chondrocytes.
This discrepancy could be explained by the higher strontium
concentrations used (3.2 ⅐ 10Ϫ3 M and 10Ϫ2 M) in his
SrCl2 (white column). Values are mean and SD (n ϭ 3). Strontium
increased the IGF-I–stimulating effect with p Ͻ 0.05.
matrix synthesis was not the result of cell proliferation, as experiment. Interestingly, strontium and calcium ranelate,
shown in bone formation,(28) because the addition of stron- but not SrCl2, increased both the PG that were released in
tium ranelate had no effect on the DNA level. Because the CM and contained in the CP of the culture. This result
SrCl2 (2 ⅐ 10Ϫ3 M) showed similar effects in the CM, the indicates that strontium ranelate promotes the synthesis of
stimulatory effect of strontium ranelate on PG production is PG capable of integrating the extracellular matrix. The
independent of the ranelic acid and strontium must be qualitative analysis of the newly synthesized PG showed
responsible for this effect. These results differ from those of that strontium ranelate (10Ϫ3 M) and calcium ranelate (10Ϫ3

306
HENROTIN ET AL.
FIG. 7. Normal human chondrocyte effects of
10Ϫ3 M strontium ranelate (-Ⅺ-) or 2 ⅐ 10Ϫ3
M
SrCl2 (-F-) compared with the controls (-E-) on
the cumulative amount of 35S-GAG (A) released
in the CM or (B) contained in the CP as a
function of the culture duration (cumulative val-
ues; means Ϯ SD). Statistical significance (A) in
the 72-h CM in which x versus y, p Ͻ 0.025, and
x versus z, p Ͻ 0.01, and (B) in the 72-h CP in
which x versus y, p Ͻ 0.01.
FIG. 8. Associative size exclusion chromatograms of 35S-labeled PG produced by normal chondrocytes cultured for 72 h in the absence (-E-)
or in the presence of 10Ϫ3 M strontium ranelate (-Ⅺ-) or 2 ⅐ 10Ϫ3 M SrCl2 (-F-). (A) CM and (B) cellular phases.
M) promoted the synthesis of high-molecular weight PG chondrocytes. Surprisingly, IGF-I stimulation of PG synthe-
(30)
complexes, which were shown to decrease in OA.
sis was increased by 10Ϫ3 M strontium ranelate treatment.
The mechanism by which extracellular strontium ranelate This effect could be consequent to chondrocyte proliferation
influenced PG synthesis is not known. However, changes in because IGF-I is required for cell proliferation.(37,38) How-
3
the extracellular ionic environment would be expected to ever, in the culture conditions used, neither H- thymidine
affect synthesis rates by altering the intracellular composi- incorporation nor DNA content was elevated in the presence
tion. It was previously shown that high extracellular Ca2ϩ of IGF-I added alone or in combination with strontium
increases intracellular free Ca2ϩ concentrations in parathy- ranelate (data not shown). Autocrine regulation of chondro-
roid cells(31) and that this effect can be mimicked by several cyte metabolism by IGF-I produced in response to a high
other divalent cations including strontium.(32) Intracellular amount of strontium also might be responsible for the PG
Ca2ϩ concentration increases could be the result of inositol increase. Indeed, recently, it was reported that a high extra-
1,4,5-triphophate receptor (InsP3R) stimulation,(33) which is cellular Ca2ϩ concentration significantly increased the se-
regulated by multiple calcium binding sites and at least four cretion of IGF-I as well as the expression of IGF-I messen-
different Ca2ϩ interaction sites.(31) The ionic strength of the ger RNA (mRNA) by osteoblastic MC3T3-E1 cells.(39) This
aqueous environment also can profoundly affect gating cal- finding could explain why strontium ranelate increased the
cium channels. Sr2ϩ and Ca2ϩ modulate the opening and IGF-I effect when it was added at a low concentration (10Ϫ9
closing of Ca2ϩ channels and in this way may affect cellular M) but not at a high concentration (10Ϫ8 M).
(34)
functions.
Strontium ranelate, calcium ranelate, and SrCl2 had sim-
Strontium could be hypothesized to be effective in re- ilar stimulating effects on TPG. Nevertheless, SrCl2 showed
versing the IGF-I–stimulating effect, because factors that no significant effect on the PG contained in the CP, and
lead to increase PG synthesis were reported as causing an calcium ranelate (10Ϫ3 M) increased the stromelysin activ-
initial decrease in cytosolic free Ca2ϩ concentration.(35,36) ity measured in the CM. Because strontium ranelate also
The effect of strontium ranelate and IGF-I added simulta- stimulated the production of type II collagen synthesis,(40) it
neously were tested on PG synthesis by normal human can be concluded that 10Ϫ3 M strontium ranelate is more

STRONTIUM RANELATE INCREASES CHONDROFORMATION
307
TABLE 3. PHYSICOCHEMICAL FORM OF THE NEWLY SYNTHESIZED PG
Kd Յ 0.13
0.13 Ͻ Kd Ͻ 0.7
Controls S12911
35S-GAG released in the culture supernatants (%)
Kd Ն 0.7
Controls
S12911
SrCl2
SrCl2
Controls
S12911
SrCl2
0–24 h
24–48 h
48–72 h
36
45
49
42
45
55
46
49
61
27
25
26
30
26
24
26
26
20
37
30
25
28
29
21
28
25
19
35S-GAG contained in the cellular phases (%)
0–24 h
24–48 h
48–72 h
29
29
37
29
36
34
28
29
37
43
48
52
40
44
52
41
50
51
28
23
11
31
20
14
31
21
12
Effects of strontium compounds on the physicochemical form of the newly synthesized PG by normal human chondrocytes. The
chomatogram was established by determining the radioactivity in each fraction and dividing into 3 groups according to a distribution
coefficient. The values are the percentage of radioactivity contained in 3 groups.
efficient in promoting cartilage formation than the other
compounds tested. Moreover, 10Ϫ3 M strontium ranelate
increased TPG in OA chondrocyte cultures and could have
beneficial effects in the prevention and the treatment of OA
lesions. This hypothesis agrees with a previous study that
showed that in strontium-induced rachitic rats the size of the
side chains of PG monomers was increased considerably in
ACKNOWLEDGMENTS
The authors thank P. Simonis, M.A. Van Simpsen, and P.
Deloffre for their valuable and continued help. This work
was supported by an unrestricted educational grant from
Servier (Courbevoie, France).
(13)
the lower part of the growth plate.
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Address reprint requests to:
Dr. Y. Henrotin
Bone and Cartilage Metabolism Research Unit
Institute of Pathology, C.H.U. Sart-Tilman Bat B23
B-4000 Lie`ge, Belgium
28. Canalis E, Hott M, Deloffre P, Tsouderos Y, Marie PJ 1996
The divalent strontium salt S 12911 enhances bone cell repli- Received in original form December 14, 1999; in revised form
cation and bone formation in vitro. Bone 18:517–523. May 17, 2000; accepted June 23, 2000.