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.код для вставкиСкачать
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: email@example.com. Submitted for publication August 14, 2002; accepted in revised form April 10, 2003. 2188 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 2190 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. 2192 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. REFERENCES 1. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum 2000;43:1916–26. CHONDROCYTE STIMULATION BY IGF-1 AND OP-1 2. Pelletier J-P, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum 2001;44:1237–47. 3. Buckwalter JA, Mankin HJ. Articular cartilage repair and transplantation. Arthritis Rheum 1998;41:1331–42. 4. Glansbeek HL, van Beuningen HM, Vitters EL, van der Kraan PM, van den Berg WB. Stimulation of articular cartilage repair in established arthritis by local administration of transforming growth factor-beta into murine knee joints. Lab Invest 1998;78:133–42. 5. Nixon AJ, Fortier LA, Williams J, Mohammed H. Enhanced repair of extensive articular defects by insulin-like growth factorI-laden fibrin composites. J Orthop Res 1999;17:475–87. 6. Fujimoto E, Ochi M, Kato Y, Mochizuki Y, Sumen Y, Ikuta Y. Beneficial effect of basic fibroblast growth factor on the repair of full-thickness defects in rabbit articular cartilage. Arch Orthop Trauma Surg 1999;119:139–45. 7. Louwerse RT, Heyligers IC, Klein-Nulend J, Sugihara S, van Kampen GP, Semeins CM, et al. Use of recombinant human osteogenic protein-1 for the repair of subchondral defects in articular cartilage in goats. J Biomed Mater Res 2000;49:506–16. 8. Guerne P-A, Blanco F, Kaelin A, Desgeorges A, Lotz M. Growth factor responsiveness of human articular chondrocytes in aging and development. Arthritis Rheum 1995;38:960–8. 9. Doré S, Pelletier J-P, DiBattista JA, Tardif G, Brazeau P, MartelPelletier J. Human osteoarthritic chondrocytes possess an increased number of insulin-like growth factor 1 binding sites but are unresponsive to its stimulation: possible role of IGF-1–binding proteins. Arthritis Rheum 1994;37:253–63. 10. Posever J, Phillips FM, Pottenger LA. Effects of basic fibroblast growth factor, transforming growth factor-beta 1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants. J Orthop Res 1995;13:832–7. 11. Chevalier X, Tyler JA. Production of binding proteins and role of the insulin-like growth factor I binding protein 3 in human articular cartilage explants. Br J Rheumatol 1996;35:515–22. 12. McQuillan DJ, Handley CJ, Campbell MA, Bolis S, Milway VE, Herington AC. Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage. Biochem J 1986;240:423–30. 13. Schalkwijk J, Joosten LAB, van den Berg WB, van Wyk JJ, van de Putte LBA. Insulin-like growth factor stimulation of chondrocyte proteoglycan synthesis by human synovial fluid. Arthritis Rheum 1989;32:66–71. 14. Lafeber FP, van der Kraan PM, Huber-Bruning O, van den Berg WB, Bijlsma JW. Osteoarthritic human cartilage is more sensitive to transforming growth factor beta than is normal cartilage. Br J Rheumatol 1993;32:281–6. 15. Lafeber FP, van Roy HL, van der Kraan PM, van den Berg WB, Bijlsma JW. Transforming growth factor-beta predominantly stimulates phenotypically changed chondrocytes in osteoarthritic human cartilage. J Rheumatol 1997;24:536–42. 16. Van Beuningen HM, van der Kraan PM, Arntz OJ, van den Berg WB. Transforming growth factor-beta 1 stimulates articular chondrocyte proteoglycan synthesis and induces osteophyte formation in the murine knee joint. Lab Invest 1994;71:279–90. 17. Reddi AH. Bone morphogenetic proteins: from basic science to clinical applications. J Bone Joint Surg Am 2001;83 Suppl:S1–6. 18. Chubinskaya S, Merrihew C, Cs-Szabo G, Mollenhauer J, McCartney J, Rueger DC, et al. Human articular chondrocytes express osteogenic protein-1. J Histochem Cytochem 2000;48:239–50. 19. Flechtenmacher J, Huch K, Thonar EJ-MA, Mollenhauer JA, Davies SR, Schmid TM, et al. Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes. Arthritis Rheum 1996;39:1896–904. 20. Loeser RF, Todd MD, Seely BL. Prolonged treatment of human osteoarthritic chondrocytes with IGF-I stimulates proteoglycan 2195 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. synthesis but not proteoglycan matrix accumulation in alginate cultures. J Rheumatol. In press. Hauselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, Aydelotte MB, et al. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J Cell Sci 1994;107:17–27. Hauselmann HJ, Masuda K, Hunziker EB, Neidhart M, Mok SS, Michel BA, et al. Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage. Am J Physiol 1996;271:C742–52. Petit B, Masuda K, D’Souza AL, Otten L, Pietryla D, Hartmann DJ, et al. Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: comparison of two distinct matrix compartments. Exp Cell Res 1996;225: 151–61. Collins DH. The pathology of articular and spinal diseases. London: Edward Arnold; 1949. p. 76–9. Muehleman C, Bareither D, Huch K, Cole AA, Kuettner KE. Prevalence of degenerative morphological changes in the joints of the lower extremity. Osteoarthritis Cartilage 1997;5:23–37. Loeser RF, Shanker G, Carlson CS, Gardin JF, Shelton BJ, Sonntag WE. Reduction in the chondrocyte response to insulinlike growth factor 1 in aging and osteoarthritis: studies in a non-human primate model of naturally occurring disease. Arthritis Rheum 2000;43:2110–20. Del Carlo M Jr, Loeser RF. Nitric oxide–mediated chondrocyte cell death requires the generation of additional reactive oxygen species. Arthritis Rheum 2002;46:394–403. Knudson CB. Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix. J Cell Biol 1993;120:825–34. Verschure PJ, van Noorden CJ, van Marle J, van den Berg WB. Articular cartilage destruction in experimental inflammatory arthritis: insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes. Histochem J 1996;28:835–57. Martel-Pelletier J, Di Battista JA, Lajeunesse D, Pelletier JP. IGF/IGFBP axis in cartilage and bone in osteoarthritis pathogenesis. Inflamm Res 1998;47:90–100. Barone-Varelas J, Schnitzer TJ, Meng Q, Otten L, Thonar EJ. Age-related differences in the metabolism of proteoglycans in bovine articular cartilage explants maintained in the presence of insulin-like growth factor I. Connect Tissue Res 1991;26:101–20. Martin JA, Ellerbroek SM, Buckwalter JA. Age-related decline in chondrocyte response to insulin-like growth factor-I: the role of growth factor binding proteins. J Orthop Res 1997;15:491–8. Messai H, Duchossoy Y, Khatib A, Panasyuk A, Mitrovic DR. Articular chondrocytes from aging rats respond poorly to insulinlike growth factor-1: an altered signaling pathway. Mech Ageing Dev 2000;115:21–37. DeGroot J, Verzijl N, Bank RA, Lafeber FPJG, Bijlsma JWJ, TeKoppele JM. Age-related decrease in proteoglycan synthesis of human articular chondrocytes: the role of nonenzymatic glycation. Arthritis Rheum 1999;42:1003–9. Van Susante JL, Buma P, van Beuningen HM, van den Berg WB,Veth RP. Responsiveness of bovine chondrocytes to growth factors in medium with different serum concentrations. J Orthop Res 2000;18:68–77. Bobacz K, Gruber R, Soleiman A, Graninger WB, Luyten FP, Erlacher L. Cartilage-derived morphogenetic protein-1 and -2 are endogenously expressed in healthy and osteoarthritic human articular chondrocytes and stimulate matrix synthesis. Osteoarthritis Cartilage 2002;10:394–401. Osborn KD, Trippel SB, Mankin HJ. Growth factor stimulation of adult articular cartilage. J Orthop Res 1989;7:35–42. Yaeger PC, Masi TL, de Ortiz JL, Binette F, Tubo R, McPherson JM. Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen 2196 and aggrecan genes in adult human articular chondrocytes. Exp Cell Res 1997;237:318–25. 39. Tsukazaki T, Usa T, Matsumoto T, Enomoto H, Ohtsuru A, Namba H, et al. Effect of transforming growth factor-beta on the insulin-like growth factor-I autocrine/paracrine axis in cultured rat articular chondrocytes. Exp Cell Res 1994;215:9–16. 40. Knutsen R, Honda Y, Strong DD, Sampath TK, Baylink DJ, LOESER ET AL Mohan S. Regulation of insulin-like growth factor system components by osteogenic protein-1 in human bone cells. Endocrinology 1995;136:857–65. 41. Yeh LC, Adamo ML, Olson MS, Lee JC. Osteogenic protein-1 and insulin-like growth factor I synergistically stimulate rat osteoblastic cell differentiation and proliferation. Endocrinology 1997; 138:4181–90.