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 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 β (TGFβ) (14), particularly with explants from the upper layer of OA cartilage (15). The disadvantage to the use of TGFβ 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 TGFβ 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 studies, 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.
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- MATERIALS AND METHODS
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 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 and30). 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 (serum-free 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 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 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 TGFβ (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.