Articular cartilage repair and growth factors in the pathogenesis of osteoarthritis

Thanks to the progress of biotechnology, in particular cloning technology, the list of growth factors that, being anabolic factors, play an important, but not fully understood role in the pathogenesis of osteoarthritis has recently been intensively expanded.

The first group of growth factors discussed below are IGFs. They are found in large quantities in blood serum and have a number of properties in common with insulin. IGF-2 is more typical for the embryonic stage of development, while IGF-1 is the dominant representative of the group in adults. Both representatives of this group act by binding to IGF type I receptors. While the function of IGF-2 remains unknown, the significance of IGF-1 has already been determined - it is able to stimulate the synthesis of proteoglycans by chondrocytes and significantly inhibit catabolic processes in articular cartilage. IGF-1 is the main anabolic stimulus for the synthesis of proteoglycans by chondrocytes, present in blood serum and synovial fluid. IGF-1 is an important factor for culturing chondrocytes in experimental models of osteoarthrosis in vitro. It is assumed that IGF-1 enters the synovial fluid from blood plasma. In addition, normal chondrocytes produce both factors - expression of IGF-1 and IGF-2 was found in the synovial membrane and cartilage of patients with osteoarthrosis. In normal cartilage, IGF-1 does not have mitogenic properties, but is able to stimulate cell proliferation in the damaged matrix, which indicates participation in reparative processes.

Biologically active substances that stimulate reparation and inhibit degradation of articular cartilage

• Insulin
• Gamma interferon
• Somatotropic hormone, androgens
• Somatomedins (IPF-1 and -2)
• TGF-beta (tissue growth factor)
• Platelet-derived growth factor
• Basic fibroblast growth factor
• EFR
• IL-1 receptor antagonist
• TNF-a-binding proteins
• Tissue inhibitors of metalloproteases
• a ₂ -macroglobulin
• ai-antitrypsin
• RG-macroglobulin
• Rg-antichymotrypsin

The actions of IGF-1 and IGF-2 are controlled by various IGF-binding proteins (IGF-BP), which are also produced by chondrocytes. IGF-BP can act as a carrier and also have IGF-blocking activity. Cells isolated from articular cartilage of patients with osteoarthrosis produce excessive amounts of IGF-BP, indicating that they block the effects of IGF. J. Martel-Pelletier et al. (1998) showed that although IGF-1 synthesis in cartilage increases in osteoarthrosis, chondrocytes respond weakly to IGF-1 stimulation. It turned out that this phenomenon is associated (at least in part) with an increase in the level of IGF-BP. IGF-BP has a high affinity for IGF and is an important biomodulator of its activity. To date, seven types of IGF-BP have been studied, and dysregulation of IGF-BP-3 and IGF-BP-4 plays an important role in osteoarthritis.

Another category of growth factors that exhibit different effects on chondrocytes includes platelet-derived growth factor (PDGF), FGF, and TGF-beta. These factors are produced not only by chondrocytes but also by activated synovocytes. FGF has both anabolic and catabolic properties depending on the concentration and condition of the articular cartilage. PDGF is involved in maintaining the homeostasis of the ECM of articular cartilage without having obvious mitogenic properties. This growth factor is known to enhance the synthesis of proteoglycans and reduce their degradation.

TGF-beta is of particular interest for its role in the pathogenesis of osteoarthritis. It is a member of the large TGF superfamily and shares functional and signaling properties with the recently discovered BMP (bone morphogenetic protein) growth factors.

TGF-beta is a pleiotropic factor: on the one hand, it has immunosuppressive properties, on the other hand, it is a chemotactic factor and a powerful stimulator of fibroblast proliferation. Unique properties of TGF-beta are the ability to inhibit the release of enzymes from various cells and significantly increase the production of enzyme inhibitors (for example, TIMP). TGF-beta is considered an important regulator of tissue damage due to inflammation. Thus, in articular cartilage tissue, TGF-beta significantly stimulates the production of matrix by chondrocytes, especially after pre-exposure to this factor. Normal cartilage is insensitive to TGF-beta. In patients with OA, TGF-β stimulates the production of aggrecan and small proteoglycans in articular cartilage.

TGF-beta is produced by many cells, particularly chondrocytes. It is released in a latent form bound to a special protein called latency-associated protein (LAP). Dissociation from this protein is accomplished by proteases, which are produced in large quantities in inflamed tissues. Apart from TGF-beta, which is produced by activated cells, stores of the latent form of this factor are an important element of TGF-beta reactivity in tissue after local injury. TGF-beta is present in significant quantities in synovial fluid, synovial membrane, and cartilage of the joint affected by osteoarthrosis. In areas of damaged tissue with inflammatory infiltrates, co-expression of TNF and IL-1 is detected, whereas in areas with fibrosis, only TGF-beta expression is detected.

Incubation of cultured chondrocytes from patients with osteoarthritis with TGF-beta causes a significant increase in proteoglycan synthesis by these cells. Stimulation of normal chondrocytes with TGF-beta causes an increase in proteoglycan synthesis only after many days of incubation. Perhaps this time is necessary for the cell phenotype to change under the influence of TGF-beta (for example, for a change in the so-called compartmentalization of proteoglycans: newly created proteoglycans are localized only around chondrocytes).

It is known that activation of growth factor synthesis, in particular TGF-beta, is an important link in the pathogenesis of renal and hepatic fibrosis, and scar formation during wound healing. Increased load on chondrocytes in vitro leads to hyperproduction of TGF-beta, while decreased proteoglycan synthesis after limb immobilization can be leveled by TGF-beta. TGF-beta induces osteophyte formation in the marginal zone of joints as a mechanism of adaptation to changes in load. IL-1, causing a moderate inflammatory process in the synovium in response to joint damage, promotes the formation of chondrocytes with an altered phenotype, which produce an excessive amount.

Repeated local injections of recombinant TGF-beta at high concentrations led to the development of osteoarthritis in C57B1 mice - the formation of osteophytes, which is characteristic of human osteoarthritis, and a significant loss of proteoglycans in the "wavy border" zone.

To understand how excess TGF-beta causes the known changes in cartilage, it is necessary to note that TGF-β exposure induces a characteristic chondrocyte phenotype with a change in the subclass of proteoglycans synthesized and disruption of the normal integration of ECM elements. Both IGF-1 and TGF-beta stimulate proteoglycan synthesis by chondrocytes cultured in alginate, but the latter also induces the so-called compartmentalization of proteoglycans. Moreover, TGF-beta was found to increase the level of collagenase-3 (MMP-13) in activated chondrocytes, which is at odds with the general idea of TGF-beta as a factor that, on the contrary, reduces the release of destructive proteases. However, it is not known whether TGF-beta-induced MMP-13 synthesis is involved in the pathogenesis of OA. TGF-beta not only stimulates the synthesis of proteoglycans, but also promotes their deposition in ligaments and tendons, increasing stiffness and reducing the range of motion in the joints.

BMPs are members of the TGF-beta superfamily. Some of them (BMP-2, BMP-7, and BMP-9) have the property of stimulating the synthesis of proteoglycans by chondrocytes. BMPs exert their effects by binding to specific receptors on the cell surface; the signaling pathways of TGF-beta and BMPs differ slightly. Like TGF-beta, BMPs signal through the serine/threonine kinase receptor complex type I and II. In this complex, the type II receptor is trans-phosphorylated and activates the type I receptor, which transmits the signal to signaling molecules called Smads. After receiving the signal, Smads are rapidly phosphorylated. It is currently known that in the BMP signaling pathway, Smads-1, -5, and -8 are phosphorylated, and in the TGF-beta signaling pathway, Smads-2 and Smad-3 are phosphorylated. Then the named Smads associate with Smad-4, which is common to the signaling pathways of all members of the TGF-beta superfamily. This fact explains the presence of cross-functions in members of the TGF-beta superfamily, as well as the phenomenon of mutual inhibition of the TGF-beta and BMP signaling pathways by competing for common components. Not long ago, another class of Smad proteins was identified, which is represented by Smad-6 and -7. These molecules act as regulators of the TGF-beta and BMP signaling pathways.

Despite the fact that the stimulating effect of CMP on proteoglycan synthesis has been known for a long time, their role in the regulation of articular cartilage function remains controversial due to the known ability of CMP to cause cell dedifferentiation, stimulate calcification and bone tissue formation. M. Enomoto-Iwamoto et al. (1998) showed that the interaction of CMP with the CMP receptor type II is necessary for maintaining the differentiated phenotype of chondrocytes, as well as controlling their proliferation and hypertrophy. According to L. Z. Sailor et al. (1996), CMP-2 maintains the phenotype of chondrocytes in culture for 4 weeks without causing their hypertrophy. CMP-7 (identical to osteogenic protein-1) maintains the phenotype of mature chondrocytes of articular cartilage cultured in alginate for a long time.

The introduction of KMP-2 and -9 into the knee joints of mice increased proteoglycan synthesis by 300%, significantly more than TGF-beta. However, the stimulating effect was temporary, and after a few days the level of synthesis returned to the initial level. TGF-beta caused a longer-term stimulation of proteoglycan synthesis, which is probably due to the autoinduction of TGF-beta and sensitization of chondrocytes to this factor.

TGF-beta is responsible for the formation of chondrophytes, which can be considered an undesirable effect of its action, KMP-2 also promotes the formation of chondrophytes, but in a different area of the articular margin (mainly in the area of the growth plate).

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Cartilage morphogenetic proteins

Cartilage morphogenetic proteins (CMP-1 and -2) are other members of the TGF-beta superfamily that are essential for the formation of cartilage tissue during limb development. Mutations in the CMP-1 gene cause chondrodysplasia. CMPs may have a more selective, cartilage-targeting profile. Although TGF-beta and CMPs can stimulate chondrocytes, they can act on many other cells, so their use for cartilage repair may be associated with side effects. Both types of CMPs are found in the cartilage of healthy and osteoarthritic joints and promote the repair of the articular cartilage ECM after enzymatic degradation, maintaining a normal phenotype.

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Synergism of growth factors

One growth factor is able to induce itself, as well as other growth factors, this interaction is finely regulated. For example, FGF together with other growth factors provides more effective reparation of articular cartilage after a traumatic defect. IGF-1 together with TGF-beta significantly induce the normal phenotype of chondrocytes when culturing them in vitro. It was demonstrated that TGF-beta prevents the production of IGF-1 and IGF-BP, and also dephosphorylates the IGF-1 receptor, stimulates IGF-1 binding. In intact mouse cartilage, the phenomenon of synergism of IGF-1 with many growth factors was found. However, the weak response of chondrocytes to IGF-1 cannot be leveled by using it in combination with other growth factors.

Interaction of anabolic and destructive cytokines

Growth factors exhibit complex interactions with IL-1. For example, preexposure of chondrocytes to FGF increases protease release after IL-1 exposure, possibly through increased IL-1 receptor expression. PDGF also stimulates IL-1-dependent protease release, but it reduces IL-1-mediated inhibition of proteoglycan synthesis. This may indicate that some growth factors can simultaneously stimulate cartilage repair and promote its destruction. Other growth factors, such as IGF-1 and TGF-β, stimulate articular matrix synthesis and inhibit IL-1-mediated articular cartilage destruction, indicating that their activity is related only to tissue repair. This interaction is independent of chondrocyte preexposure to IL-1. Interestingly, the kinetics of the effects of IL-1 and TGF-beta may be different: the ability of TGF-beta to suppress articular cartilage degradation is attenuated by its slow action on TIMP mRNA. On the other hand, an increase in hNOC and NO levels is observed in the absence of TGF-beta. Given the NO-dependence of the suppressive effect of IL-1 on proteoglycan synthesis by chondrocytes, it may explain why we observe a significantly stronger counteraction of TGF-beta to IL-1-dependent inhibition of proteoglycan synthesis compared to proteoglycan degradation in vivo.

In a study in mice injected intra-articularly with IL-1 and growth factors, it was demonstrated that TGF-beta significantly counteracts IL-1-mediated inhibition of articular cartilage proteoglycan synthesis, whereas CMP-2 is incapable of such counteraction: its stimulatory potential was completely inhibited by IL-1 even at high concentrations of CMP-2. Notably, in the absence of IL-1, CMP-2 stimulated proteoglycan synthesis much more intensely than TGF-beta.

In addition to its effect on proteoglycan synthesis, TGF-beta also significantly affects IL-1-induced reduction in cartilage proteoglycan content. It is possible that proteoglycan content decreases or increases depending on the relative concentration of IL-1 and TGF-beta. Interestingly, the above-described counteraction of IL-1 and TGF-beta was observed in the thickness of cartilage, but this phenomenon was not observed near chondrophytes at the edges of articular surfaces. Chondrophyte formation is induced by TGF-β, which affects chondrogenic cells in the periosteum, causing the development of chondroblasts and proteoglycan deposition. Apparently, these chondroblasts are not sensitive to IL-1.

H. L. Glansbeek et al. (1998) studied the ability of TGF-beta and KMP-2 to counteract the suppression of proteoglycan synthesis in the joints of mice with zymosan-induced arthritis (i.e., in a model of "pure" IL-1-induced inflammation). Intra-articular administration of TGF-beta significantly counteracted the suppression of proteoglycan synthesis caused by inflammation, while KMP-2 was virtually unable to counteract this IL-1-dependent process. Repeated injections of TGF-β into the knee joint of the studied animals significantly stimulated proteoglycan synthesis by chondrocytes, contributed to the preservation of existing proteoglycans in cartilage depleted by inflammation, but did not suppress the inflammatory process.

When studying the proteoglycan-synthesizing function of chondrocytes using experimental models of osteoarthrosis in animals, an increase in the content and stimulation of the synthesis of proteoglycans in the early stages of OA has always been noted, in contrast to inflammatory models, in which significant inhibition of synthesis (IL-1-dependent process) is observed. Increased activity of anabolic factors, in particular growth factors, which is observed in osteoarthrosis, neutralizes the effect of such suppressor cytokines as IL-1. Among the growth factors, TGF-beta is of the greatest importance; KMP-2 is unlikely to play a significant role in this process. Although IGF-1 is able to stimulate proteoglycan synthesis in vitro, this property is not observed in vivo with local application of IGF-1. This may be due to the fact that the endogenous level of this growth factor is optimal. In later stages of osteoarthritis, signs of inhibition of proteoglycan synthesis appear, which is probably associated with the dominant action of IL-1 and the inability of growth factors to counteract it due to decreased activity.

Analysis of growth factor expression in STR/ORT mice with spontaneous osteoarthritis demonstrated increased mRNA levels of TGF-β and IL-1 in damaged cartilage. It should be noted that activation of TGF-β from the latent form is an important element of tissue repair. Understanding the role of TGF-β is complicated by the results of a study of TGF-β type II receptor expression in ACL rabbits. Immediately after induction of osteoarthritis, decreased levels of these receptors were detected, indicating insufficient TGF-β signaling. Interestingly, TGF-β receptor type 11-deficient mice showed signs of spontaneous osteoarthritis, which also indicates an important role of TGF-β signaling in the deterioration of cartilage repair and the development of osteoarthritis.

The absolute content of growth factors in the joints of patients with rheumatoid arthritis or osteoarthrosis may indicate their possible role in the pathogenesis of these diseases. However, despite the fact that high concentrations of growth factors are found in joints with osteoarthrosis and rheumatoid arthritis, the nature of the degradation and reparation processes in both diseases is completely different. Probably, there are other, as yet unidentified factors that play a major role in the pathogenesis of these diseases, or other aspects of the phenomena studied determine the course of degradation and reparation processes in joint tissues (for example, the expression of certain receptors on the surface of chondrocytes, soluble receptors that bind proteins, or an imbalance of anabolic and destructive factors).