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The combination of insulin-like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 8, August 2003, pp 2188–2196
DOI 10.1002/art.11209
© 2003, American College of Rheumatology
The Combination of Insulin-Like Growth Factor 1 and
Osteogenic Protein 1 Promotes Increased Survival of and
Matrix Synthesis by Normal and Osteoarthritic Human
Articular Chondrocytes
Richard F. Loeser, Carol A. Pacione, and Susan Chubinskaya
improved survival, to 87 ⴞ 2% for OA cells and 95 ⴞ 1%
for normal cells. Cell proliferation was noted only in the
IGF ⴙ OP group; this was significant for both normal
and OA cells (⬃2-fold increase in DNA levels). Matrix
production, assessed by particle exclusion and by proteoglycan accumulation, was greatest in the cells treated
with IGF ⴙ OP in both normal and OA cultures. When
proteoglycan levels were corrected for cell numbers (␮g
proteoglycan/ng DNA), a significant increase over control was noted with OP-1 alone and IGF ⴙ OP, but not
IGF-1 alone, in both normal and OA cultures, with the
greatest levels in the combination group (3-fold increase
over control).
Conclusion. OP-1 was more potent than IGF-1 in
stimulating proteoglycan production in both normal
and OA cells. However, the best results were obtained
with the combination, suggesting that combined therapy
with IGF-1 and OP-1 may be an effective strategy for
treating OA cartilage damage.
Objective. Although growth factor therapy could
be an attractive method for stimulating the repair of
damaged cartilage matrix, there is evidence that with
aging and/or with the development of osteoarthritis
(OA), articular chondrocytes may become unresponsive
to growth factor stimulation. The aim of the current
study was to compare the ability of insulin-like growth
factor 1 (IGF-1) and osteogenic protein 1 (OP-1), alone
and in combination, to stimulate human normal and OA
chondrocytes in culture.
Methods. Chondrocytes isolated by enzymatic digestion of cartilage obtained from subjects undergoing
knee replacement for OA (n ⴝ 6) or from normal ankle
joints of tissue donors (n ⴝ 7) were cultured in alginate
beads in serum-free medium and treated for 21 days
with 100 ng/ml IGF-1, 100 ng/ml OP-1, or both. Controls
were treated with vehicle alone. The cultures were
evaluated for cell survival, cell number by DNA analysis,
matrix production by particle exclusion assay, and level
of accumulated proteoglycan by dimethylmethylene blue
assay.
Results. After 21 days in serum-free alginate
culture, survival of cells from OA cartilage was 65 ⴞ 2%
(mean ⴞ SEM), while survival of cells from normal
cartilage was significantly greater (82 ⴞ 3%). Treatment
with either IGF-1 or OP-1 alone minimally improved
survival, while the combination IGF ⴙ OP significantly
An imbalance between the activity of anabolic
and catabolic pathways within the cartilage matrix results in the destruction and loss of articular cartilage
during the development of osteoarthritis (OA) (1,2).
The articular chondrocyte is the only cell type present in
cartilage and is therefore responsible for both matrix
production and destruction. The balance of these processes depends on the local activity of regulatory factors
including growth factors and cytokines. Because of their
ability to stimulate chondrocyte anabolic activity, and in
some cases inhibit catabolic activity, growth factors may
be useful agents to combat the loss of the cartilage
matrix in arthritis.
Although some limited success of growth factor
therapy has been demonstrated in animal models of
arthritis or cartilage damage (3–7), there is a lack of data
Supported by NIH grants AG-16697 (to Dr. Loeser) and
AG-47654 (to Dr. Chubinskaya).
Richard F. Loeser, MD, Carol A. Pacione, BS, Susan Chubinskaya, PhD: Rush Medical College of Rush–Presbyterian–St.
Luke’s Medical Center, Chicago, Illinois.
Address correspondence and reprint requests to Richard F.
Loeser, MD, Rheumatology, Rush–Presbyterian–St. Luke’s Medical
Center, 1725 W. Harrison, Suite 1017, Chicago, IL 60612. E-mail:
rloeser@rush.edu.
Submitted for publication August 14, 2002; accepted in
revised form April 10, 2003.
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CHONDROCYTE STIMULATION BY IGF-1 AND OP-1
from human cartilage to fully assess the feasibility of
growth factor therapy. A potential reduction in the
capacity of chondrocytes from older adult humans to
respond to growth factor stimulation may be a major
limiting factor in the use of growth factors to treat matrix
damage (8). Studies have shown that human OA chondrocytes may lack an anabolic response to insulin-like
growth factor 1 (IGF-1) (9–11), a growth factor which is
native to cartilage and which is normally the major
chondrocyte stimulator of proteoglycan synthesis in serum and synovial fluid (12,13). However, OA chondrocytes do not appear to be unresponsive to all growth
factors. Studies have shown that OA cartilage explants
may actually be more responsive than normal cartilage
when stimulated with transforming growth factor ␤
(TGF␤) (14), particularly with explants from the upper
layer of OA cartilage (15). The disadvantage to the use
of TGF␤ as growth factor therapy for repair of cartilage
damage is that it also stimulates osteophyte formation
(4,16).
Osteogenic protein 1 (OP-1), also known as bone
morphogenetic protein 7, is an anabolic growth factor
which is a member of the TGF␤ superfamily (17) and
which is expressed in cartilage (18). OP-1 has been
shown to be a very potent stimulator of chondrocyte
proteoglycan and collagen synthesis (19). Thus, it has
potential as a cartilage repair factor, but its ability to
stimulate matrix production by OA chondrocytes has
received limited attention.
In a recent study, investigators at our laboratory
found that when chondrocytes isolated from human OA
cartilage were stimulated with IGF-1, an increase in
sulfate incorporation (as a measure of proteoglycan
synthesis) could be detected after 7–10 days of culture,
but significant matrix accumulation of proteoglycans
could not be detected (20). In contrast to IGF-1, OP-1
treatment stimulated proteoglycan matrix accumulation.
The objective of the present study was to measure and
compare the response of chondrocytes isolated from
normal and OA cartilage to IGF-1 and OP-1, alone and
in combination. The combination was included to test
the hypothesis that chondrocytes would respond better
to a combination of growth factors as compared with
either growth factor alone.
For these studies, chondrocytes were cultured in
suspension in alginate beads in order to maintain the
differentiated chondrocyte phenotype (21) and to quantify effects of the growth factors on cell survival, proliferation, and matrix production in relatively long-term
cultures. A 21-day culture period was chosen based on
previous reports (22,23) and our own preliminary stud-
2189
ies, which demonstrated that significant matrix accumulation could be measured by 21 days of alginate culture
and that further culture was unlikely to change the
overall results.
MATERIALS AND METHODS
Chondrocyte isolation and culture. Normal cartilage
was obtained from the ankle joints of 7 tissue donors, through
the assistance of the Regional Organ Bank of Illinois. The
donors had no known history of arthritis. Each joint was
graded on a modified Collins scale (24) for gross evidence of
damage, as described (25). Only normal or nearly normal
tissue (grade 0 or 1) was used. The mean ⫾ SD age of the
donors was 45 ⫾ 8 years (range 20–76). OA cartilage was
obtained from tissue removed at the time of total knee
replacement surgery (n ⫽ 6). This tissue was kindly provided
by the Department of Orthopaedic Surgery at Rush–
Presbyterian–St. Luke’s Medical Center, with Institutional
Review Board approval. The average age of the OA patients
was 66 ⫾ 11 years (range 51–78), which was higher than the age
of the normal donors, but the difference did not quite reach
statistical significance (P ⫽ 0.09).
Cartilage was dissected from the joints, with care taken
to avoid underlying bone or tissue from osteophytes. Cartilage
slices were digested in Dulbecco’s modified Eagle’s medium
(DMEM)/Ham’s F-12 medium (1:1) containing 0.2% Pronase
(Calbiochem, San Diego, CA) in an incubator with continuous
agitation for 1 hour, and then overnight with 0.025% collagenase P (Roche, Indianapolis, IN) in DMEM/Ham’s F-12
supplemented with 5% fetal bovine serum. After isolation, the
cells were counted. The initial viability prior to culture was
assessed using trypan blue dye exclusion and was determined
to be ⬎90%. The cells were resuspended at 2 ⫻ 106/ml in
sodium alginate. Alginate beads were produced as previously
described (26), resulting in ⬃20,000 cells per bead.
Alginate beads were cultured, at 8 beads per well in
24-well plates, in serum-free DMEM/Ham’s F-12 (1:1; 0.5
ml/well). All media were supplemented with 1% mini-ITS⫹,
which contains 5 nM insulin (“mini”-dose insulin so that the
IGF-1 receptor is not stimulated), 2 ␮g/ml transferrin, 2 ng/ml
selenous acid, 25 ␮g/ml ascorbic acid, and bovine serum
albumin/linoleic acid at 420/2.1 ␮g/ml (26). IGF-1–treated
wells received 100 ng/ml of recombinant human IGF-1 (a gift
from Chiron Corp., Emeryville, CA), OP-1 treated wells
received 100 ng/ml recombinant human OP-1 (provided by
Stryker Biotech, Hopkinton, MA), and the combination treatment wells received 100 ng/ml of each growth factor. Triplicate
wells were used for each condition. Medium was changed every
48 hours, with the addition of fresh growth factor to the treated
wells.
For comparison with growth factor treatments, some
initial experiments also included cultures maintained in
DMEM/Ham’s F-12 supplemented with 10% fetal bovine
serum. After 14–21 days in serum-containing cultures, significant numbers of chondrocytes were noted to have migrated out
of the alginate beads, establishing a monolayer of cells at the
bottom of the culture wells. This correlated with reduced cell
numbers in the beads from serum-containing cultures when
analyzed on day 21 (data not shown). No cells were observed
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LOESER ET AL
on the bottom of any of the wells containing beads cultured in
serum-free media with or without growth factors. Because of
the loss of cells in alginate beads cultured in media with serum,
these results were not included with the growth factor results.
Survival assay. Cell survival was measured as previously described in detail (27), using calcein AM and ethidium
bromide homodimer 1 (Molecular Probes, Eugene, OR). Live
cells emit a green fluorescence after metabolizing calcein by a
ubiquitous intracellular esterase, while ethidium bromide homodimer 1 enters cells upon the compromise of plasma
membrane integrity after cell death and stains nuclear DNA
red. At least 100 cells were counted in triplicate for each data
point.
Matrix assessment by particle exclusion assay. The
pericellular matrix retained by the cultured cells was visualized
using a particle exclusion assay as previously described (22,28).
Briefly, the alginate was solubilized with sodium citrate, and
the cells were pelleted by centrifugation, resuspended in
DMEM, and then spun down to the culture surface of a
multiwell plate, by cytospinning. A suspension of formalinfixed erythrocytes was added and allowed to settle for ⬃10
minutes. The presence of a pericellular matrix excludes the
erythrocytes from the cell membrane. The cells were visualized
and photographed with an inverted phase-contrast microscope.
Dimethylmethylene blue (DMB) assay for proteoglycan production. After 21 days of culture, the medium was
removed and the alginate beads (8 per well) were collected and
placed in microfuge tubes. The beads were dissolved in sodium
citrate and centrifuged to separate the cell pellet (cells and
cell-associated matrix) from the remainder of the matrix. Cell
pellets were treated with 0.02% sodium dodecyl sulfate followed by digestion with proteinase K (125 ␮g/ml; Calbiochem)
prior to the DMB assay and DNA analysis. Samples of digested
cell pellets and samples of alginate matrix were used in the
DMB assay performed as described (22), using bovine nasal
septum D1 proteoglycan standard (provided by Dr. Eugene
Thonar, Rush Medical College). In the DNA assay, PicoGreen
(Molecular Probes) was used instead of Hoechst dye.
Statistical analysis. The statistical significance of results was determined by analysis of variance, using StatView
5.0 software (SAS Institute, Cary, NC).
RESULTS
Stimulation of cell survival and proliferation by
IGF-1 and OP-1. Mean ⫾ SEM cell survival on day 21 of
serum-free culture was significantly greater (P ⫽ 0.005)
in cultures initiated with chondrocytes isolated from
normal cartilage (82 ⫾ 3%) than in cultures of cells from
OA cartilage (65 ⫾ 2%). Compared with untreated
controls, either IGF-1 or OP-1 treatment resulted in a
slight increase in survival of normal cells, to 88 ⫾ 2%
(P ⫽ 0.09) with IGF-1 and 90 ⫾ 2% (P ⫽ 0.01) with
OP-1 (Figure 1A). The best survival (95 ⫾ 1%; P ⫽
0.0003) was noted in cultures treated with the combination of IGF ⫹ OP-1. In OA cultures, IGF-1 alone did
not improve survival; however, OP-1 improved survival
to 73 ⫾ 3% (P ⫽ 0.05). Similar to the results with normal
Figure 1. Survival of normal (A) and osteoarthritic (OA) (B) chondrocytes after 21 days of culture in serum-free media with or without
insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1).
Chondrocytes were cultured in alginate beads in serum-free media
(control [CNTL]) or serum-free media supplemented with 100 ng/ml
IGF-1, 100 ng/ml OP-1, or both. Cell survival was measured using
fluorescent dyes as described in Materials and Methods. Values are the
mean and SEM from cultures established from normal tissue (n ⫽ 7)
or OA tissue (n ⫽ 6).
cells, the best survival of OA cells (87 ⫾ 2%) was found
with the combination of IGF-1 ⫹ OP-1; with this
treatment, survival was significantly better than survival
in serum-free controls (P ⬍ 0.0001) or with IGF-1 (P ⫽
0.0001) or OP-1 (P ⫽ 0.005) alone (Figure 1B).
As detailed in Materials and Methods, evaluation
of cell survival required incubating cells with calcein,
which is converted by live cells to a green fluorescent
product retained in the cell. When the cells treated for
21 days with the IGF-1 ⫹ OP-1 combination were
incubated with calcein and then observed under a fluorescent microscope, a pattern of green fluorescent cells
grouped in a circular arrangement was noted (Figure 2).
Rarely, small groups of cells were observed with OP-1
alone, while mainly individual cells were seen in the
control and IGF-1–treated cultures. The cell clusters in
the IGF-1 ⫹ OP-1–treated cultures were a consistent
finding that was observed with cells from both normal
and OA cartilage. When cultures were observed at
earlier time points, the cell cluster pattern was not seen
until approximately day 14 of culture.
The arrangement of the cells in clusters suggested cell proliferation had occurred. This was confirmed by the results of the DNA analysis. DNA levels
correlated with survival results in that a higher DNA
content was found in normal compared with OA cultures
(mean ⫾ SEM 8.3 ⫾ 1 versus 5.6 ⫾ 1 ␮g/8 beads; P ⫽
0.05). In separate experiments using agents that induce
cell death, we have noted that DNA levels measured
with PicoGreen correlate very closely with the cell
survival measures when the cells have been dead for ⬎24
hours (Loeser RF, et al: unpublished observations). This
is probably because PicoGreen binds mainly to double-
CHONDROCYTE STIMULATION BY IGF-1 AND OP-1
2191
Figure 2. Arrangement of OA chondrocytes after 21 days of culture in alginate. Chondrocytes were cultured as
described in Figure 1. After incubation of alginate beads with calcein AM and ethidium bromide homodimer 1,
the beads were dissolved in sodium citrate. A sample was removed and immediately photographed using
fluorescent microscopy. Live cells emit a strong green cytoplasmic fluorescence, while dead cells exhibit red
nuclear fluorescence. See Figure 1 for definitions.
stranded DNA. After the cells have died, DNA is
degraded and unwinds to single-stranded fragments that
would not be detected by the PicoGreen.
Compared with serum-free controls, normal cells
had ⬎2.2-fold higher DNA levels after 21 days of
treatment with IGF-1 ⫹ OP-1 (Figure 3A). This result
suggested not only improved survival but also proliferation of cells, since in these cultures survival was increased by only 13% compared with serum-free controls
(see above). A similar 2.3-fold higher DNA level was
noted in cultures of OA cells treated with the combination of growth factors (Figure 3B). Baseline DNA levels
on day 1 of culture were measured in cells from 3 OA
patients, and the mean ⫾ SEM level was found to be
5.6 ⫾ 0.4 ␮g/8 beads. After 21 days of treatment with
IGF-1 ⫹ OP-1, the mean DNA level in cells from the
same subjects was 12.4 ⫾ 0.7 ␮g/8 beads, consistent with
a 2.2-fold increase in cell numbers. This provided further
support for the notion that IGF-1 ⫹ OP-1 induced
proliferation.
Stimulation of matrix production by IGF-1 and
OP-1. After 21 days of culture, the alginate beads
containing either normal or OA chondrocytes that were
treated with the combination of IGF-1 ⫹ OP-1 were
noticeably larger than the controls or the beads treated
Figure 3. Cell numbers in 21-day cultures of normal (A) and OA (B)
chondrocytes. Alginate bead cultures of chondrocytes, treated as
described in Figure 1, were evaluated for cell numbers by an assay for
total DNA. Values are the mean and SEM from cultures established
from normal tissue (n ⫽ 7) or OA tissue (n ⫽ 6). See Figure 1 for
definitions.
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LOESER ET AL
Figure 4. Alginate beads after 21 days of culture. Normal articular chondrocytes were cultured as described in
Figure 1, with the addition of a set of beads cultured in media containing 10% fetal bovine serum. Beads were
removed from the culture wells at the end of the 21-day culture period and immediately photographed. See
Figure 1 for definitions.
with either growth factor alone (Figure 4). Unlike the
findings with the other cultures, the beads treated with
the combination of growth factors had an irregular
surface with numerous small protrusions and bulges
which, when viewed under the microscope, were noted
to contain cells and cell-associated matrix. When visualized using the particle exclusion assay, the pericellular
matrix was largest in cultures treated with OP-1 or the
combination of IGF-1 ⫹ OP-1 (Figure 5). In the combination treatment group, clusters of cells with an associated matrix were observed. These findings were similar
in cultures of normal and OA chondrocytes.
Stimulation of matrix proteoglycan production
by IGF-1 and OP-1. When normal chondrocyte cultures
were incubated with OP-1 or the combination of IGF1 ⫹ OP-1, the total amount of proteoglycan produced
and retained in the alginate beads after 21 days of
culture was significantly greater than in control cultures
or IGF-1–treated cultures (proteoglycan level with OP-1
treatment ⬎3-fold that in controls; proteoglycan level
with IGF-1 ⫹ OP-1 treatment ⬎6-fold that in controls)
(Figure 6A). A similar increase in the total amount of
proteoglycan was noted in OA cultures treated with
OP-1 (2.7-fold) or IGF-1 ⫹ OP-1 (5.5-fold) (Figure 6C).
Although IGF-1 alone did not increase proteoglycan
levels, there was significantly more proteoglycan in
beads with OA cells treated with IGF-1 ⫹ OP-1 compared with OP-1 alone (P ⬍ 0.0001).
When values were normalized for the differences
in cell numbers by using the DNA values, treatment with
either OP-1 or the combination of IGF-1 ⫹ OP-1
resulted in a significant increase in the amount of
proteoglycan per cell in both normal and OA cultures,
compared with control cultures and those treated with
IGF-1 alone (Figures 6B and D). The OA cultures
treated with IGF-1 ⫹ OP-1 also had significantly more
proteoglycan per cell than cultures treated with OP-1
alone (P ⫽ 0.05). The total amount of proteoglycan
deposited in the alginate beads after treatment with
Figure 5. Cell-associated matrix after 21 days of culture. Chondrocytes isolated from normal cartilage were cultured in alginate beads as
described in Figure 1. At the end of the culture period, the beads were
dissolved in sodium citrate and the cells collected by centrifugation.
Cells were cytospun and then incubated with fixed erythrocytes as
described in Materials and Methods. A representative sample was
photographed using an inverted microscope with phase contrast. The
cell-associated matrix can be seen excluding the erythrocytes from the
chondrocyte plasma membrane. See Figure 1 for definitions.
CHONDROCYTE STIMULATION BY IGF-1 AND OP-1
Figure 6. Chondrocyte proteoglycan production after 21 days of
alginate culture. Chondrocytes isolated from normal cartilage (A and
B) and from OA cartilage (C and D) were cultured as described in
Figure 1. The total amount of proteoglycan deposited in the cellassociated and further-removed matrix was measured as described in
Materials and Methods, and the amount of DNA in the cell pellets was
measured. Values are the mean and SEM from cultures established
from normal tissue (n ⫽ 7) or OA tissue (n ⫽ 6). See Figure 1 for
definitions.
IGF-1 ⫹ OP-1 was greater in beads with chondrocytes
from normal cartilage (mean ⫾ SEM 152 ⫾ 12 ␮g/8
beads) compared with beads with OA chondrocytes
(110 ⫾ 11 ␮g/8 beads). However, after correction for
differences in cell numbers by the DNA assay, the
amount of proteoglycan per cell was similar in normal
and OA cultures (8.4 ⫾ 0.6 and 9 ⫾ 0.8 ␮ g
proteoglycan/ng DNA, respectively).
DISCUSSION
Under the conditions used in the present study,
adult human articular chondrocytes, isolated from either
normal or osteoarthritic cartilage, responded better to
OP-1 than to a similar concentration of IGF-1. When
the 2 growth factors were combined, the effects appeared to be different from the effect of either growth
factor used alone. The combination of the 2 growth
factors stimulated cell proliferation and reduced cell
death in 3-week serum-free cultures. Importantly, the
stimulation of proliferation did not result in decreased
matrix production, so the total amount of proteoglycan
matrix produced was greatest in the combination group.
This was the case for cells from both normal and OA
cartilage. In fact, there was no difference between
normal and OA cultures in growth factor–stimulated
proteoglycan production after correction for differences
in cell numbers. Since OA cartilage is characterized by a
2193
loss of matrix and, at least in advanced stages, a loss of
cells due to cell death, the results suggest that combined
treatment of damaged cartilage with IGF-1 and OP-1
may be a useful strategy to develop further.
The lack of a stimulatory effect of IGF-1 on
matrix production in cultures of OA chondrocytes was
consistent with the results of previous studies (9,10,26).
The finding that IGF-1 did not stimulate proteoglycan
production by chondrocytes from normal adult human
cartilage was a bit surprising in light of the accepted
notion that IGF-1 is an important anabolic factor in
cartilage (for review, see refs. 29 and 30). In the present
study, we examined chondrocytes isolated from normal
adult human tissue donors with an average age of 45
years, and the cells were cultured under serum-free
conditions in alginate. The reasons for a poor response
to IGF-1 could be related to the source of the cells
(adult human ankle) or the culture conditions (serumfree alginate); each of these possibilities is discussed
below.
Changes in chondrocytes with aging can affect
growth factor responsiveness. An age-related decline in
IGF-1 response has been noted in bovine (31), rat
(32,33), and monkey chondrocytes (26), although there
is a lack of data regarding human chondrocytes. A
previous study demonstrated an age-related decrease in
the mitogenic response to IGF-1 (8), and the ability of
10% serum to stimulate sulfate incorporation by human
chondrocytes in explant culture was shown to decrease
with donor age (34). In the present study, we did not
evaluate enough samples from donors of different ages
to determine if age was responsible for the poor response to IGF-1. Nevertheless, the poor response to
IGF-1 found in the present study is important since
those most likely to have cartilage matrix damage that
could benefit from growth factor therapy are older
adults.
Many of the early in vitro studies that documented IGF-1 stimulation of chondrocyte proteoglycan
production used cells from young animals, often cultured in media with serum. However, a recent study
showed that young bovine chondrocytes in serum-free
alginate culture responded to 100 ng/ml IGF-1 with
increased sulfate incorporation, equal to that obtained
with 10% serum (35). In that study, addition of IGF-1 to
10% serum resulted in a 2-fold increase in sulfate
incorporation compared with addition of IGF-1 to
serum-free media. Our study differs in that, rather than
measuring short-term sulfate incorporation as an indication of IGF-1 response, we measured the total amount
of proteoglycan produced and retained in the matrix
2194
during a 3-week culture period. We did not add serum to
the IGF-1–treated cultures because we wished to evaluate the response of the cells under serum-free conditions
and because we found that in long-term alginate cultures, serum stimulates migration of cells out of the
beads. In one experiment in which we used cells from a
65-year-old donor and moved the beads to fresh plates
every week (which prevents loss of cells by migration),
we did not find a significant increase in proteoglycan
levels in cultures treated with IGF-1 in 10% serum
compared with 10% serum alone as a control (Loeser
RF, et al: unpublished observation).
It is unlikely that the joint site (ankle) was the
reason for the lack of an IGF-1 response in normal cells
since previous animal studies have used cartilage from
various sites, such as the commonly used metatarsophalangeal joints of cows (fetlock joint). In an experiment
using chondrocytes isolated from the knee cartilage of
one of the same tissue donors from whom an ankle
specimen was obtained, there was a similar lack of IGF-1
response (data not shown). We also do not believe the
lack of IGF-1–stimulated proteoglycan accumulation
was due to a lack of functional IGF-1 receptors. We have
been able to detect significant stimulation of the Akt
protein kinase by IGF-1 in the same system used for the
present studies (Loeser RF, et al: unpublished observations). However, this finding only confirms that the
IGF-1 receptor was active and does not provide evidence
needed to judge the signaling required to stimulate
proteoglycan production. Akt is known to be involved in
cell survival signaling, but it is not known if it is part of
the signaling pathway that regulates proteoglycan synthesis, since this pathway has not been fully defined.
OP-1 has previously been shown to be a potent
stimulator of proteoglycan and collagen synthesis in
human chondrocytes in short-term alginate culture (19).
The earlier study used only chondrocytes from normal
cartilage, and the age of the oldest donor was 44 years.
Findings of the present study demonstrate that chondrocytes from healthy older donors (up to 76 years) respond
to OP-1, and, importantly, these findings provide extensive data showing that chondrocytes from human OA
cartilage also respond to OP-1 in alginate culture. In
fact, the amount of total proteoglycan produced per cell
in response to OP-1 was similar between cells from
normal and those from OA cartilage. These results
confirm and extend the results of a recent study using
human OA cells in monolayer (36) and a pilot study of
treatment with OP-1 alone in human OA chondrocytes
in alginate (20).
Perhaps the most important and novel finding of
LOESER ET AL
the present study was that when IGF-1 and OP-1 were
used together, cell proliferation was noted and a greater
increase in proteoglycan production was found in both
normal and OA cultures. This finding is of particular
interest given the result that IGF-1 by itself did not
stimulate proteoglycan levels. The mechanism behind
the response to combined growth factor treatment is not
clear. Studies have shown that either epidermal growth
factor (37), fibroblast growth factor (37), or TGF␤
(38,39) can modulate the response of chondrocytes to
IGF-1. The combination of OP-1 with IGF-1 has not
been previously studied with chondrocytes. In bone cells,
OP-1 has been shown to modulate the expression of
components of the IGF-1 regulatory system, which
include the IGF binding proteins (IGFBPs) and IGFBP
proteases. OP-1 was found to increase expression of
IGFBP-3 and IGFBP-5, while decreasing IGFBP-4 and
the IGFBP-5 protease (40). Combined IGF-1 and OP-1
treatment of rat osteoblastic cells stimulated OP-1–
induced proliferation (41). If OP-1 reduces expression of
an inhibitory IGFBP or increases a stimulatory IGFBP,
it could improve the IGF-1 response. The IGFBPs were
not measured in the present study since it is still not
clear which would be inhibitory or stimulatory to IGF-1
action in cartilage, which would make it difficult to
interpret results.
In summary, the findings of the current study
show that, based on in vitro assessment of matrix
proteoglycan accumulation, human chondrocytes isolated from adult tissue donors without arthritis as well as
chondrocytes from osteoarthritic cartilage respond to
OP-1 but not to IGF-1. However, the best results in
terms of cell survival and total matrix production are
seen when the growth factors are combined. Further
work is needed to better understand the mechanisms for
the effect of combined IGF-1 and OP-1 and to determine if the combination of IGF-1 and OP-1 will promote
cartilage repair in vivo.
ACKNOWLEDGMENTS
We would like to thank the Regional Organ Bank of
Illinois and Dr. Arkady Margulis for providing human donor
tissues, and the Department of Orthopaedic Surgery at Rush–
Presbyterian–St. Luke’s Medical Center for OA tissues. We
also thank Dr. David Rueger of Stryker Biotech for providing
OP-1, and Dr. Klaus Kuettner for helpful discussions.
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new therapeutic targets. Arthritis Rheum 2001;44:1237–47.
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