Transforming growth factor beta superfamily members: role in cartilage modeling

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.     

Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone

We examined immunohistochemically the fracture repair process in rat tibial bone using antibodies to PCNA, BMP2, TGF-beta 1,-2,-3, TGF-beta R1,-R2, bFGF, bFGFR, PDGF, VEGF, and S-100. The peak level of cell proliferation as revealed by PCNA labelling appeared first in primitive mesenchymal cells and inflammatory cells at the fracture edges and neighboring periosteum at 2-days after fracture, followed by the peaks of periosteal primitive fibroblasts and chondroblasts, which appeared at fracture edges at 3- and 4-days after fracture, respectively. BMP2 was weakly positive in primitive mesenchymal cells, osteoblasts and chondroblasts. At 3-days post-fracture, periosteal osteoblasts produced osteoid tissue and callus with marrow spaces lined by osteoblasts and osteoclasts, and all primitive mesenchymal cells and osteoblasts were positive for TGF-beta 1,-2,-3, and TGF-beta R1,-R2. They were also positive for vascular growth factors bFGF, FGFR and PDGF, but negative for VEGF, and the peak of PCNA labelling of vascular endothelial cells in the marrow space was delayed to 4-days after fracture. Chondroblasts at fracture edges produced hypertrophic chondrocytes at 5-days after fracture and they were positive for TGF-beta 1,-2,-3, and TGF-beta R1,-R2. Primitive chondroblasts were positive for vascular growth factors VEGF as well as bFGF, FGFR, and the peak of PCNA labelling of vascular endothelial cells in the cartilage was at 5-days after fracture. Hypertrophic chondrocytes were also positive for these growth factors but negative for bFGF and bFGFR. S-100 protein-induced calcification was only positive on chondroblasts and hypertrophic chondrocytes. At 7-days after fracture, bone began to be formed from the cartilage at fracture edges, by a process similar to bone formation in the growth plate. Enchondral ossification established a bridge between both fracture edges and periosteal membranous ossification encompassed the fracture site like a sheath at 14 day after fracture. Our study of fracture repair of bone indicates that this process is complex and occurs through various steps involving various growth factors.     

Immunolocalization of IL-17A, IL-17B, and their receptors in chondrocytes during fracture healing.

Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in the epiphyseal growth plate.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013. © 2012 Wiley Periodicals, Inc.     

Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo

Although it has been clearly established that basic fibroblast growth factor (FGF) is a potent mitogen for chondrocytes in vitro, there is little evidence that it can stimulate this cell type in vivo. In an effort to address this problem, we examined the effect of an intraarticular administration of basic FGF. Alzet osmotic pumps delivering the mitogen to the site of injury promotes the healing of intra-chondrial lesions by stimulating chondrocyte proliferation and the formation of extracellular matrix. The observation that chronic infusions of basic FGF can elicit a repair response at the site of injury suggests that this growth factor may have therapeutic applications that extend beyond its capacity to induce neovascularization. The results also suggest that one of the ways that the perichondrium mediates cartilage repair may be by the local production of FGF-like mitogens.     

Peptide growth factors and their interactions during chondrogenesis

Peptide growth factors have been implicated in three aspects of cartilage growth and metabolism; the induction of mesoderm and differentiation of a cartilaginous skeleton in the early embryo, the growth and differentiation of chondrocytes within the epiphyseal growth plates leading to endochondral calcification, and the processes of articular cartilage damage and repair. Three peptide growth factor classes have been strongly implicated in these processes, the fibroblast growth factor family (FGF), the insulin-like growth factors (IGFs) including insulin, and transforming growth factor-β (TGF-β) and related molecules. Each of these peptide groups are expressed in the early embryo. Basic FGF, TGF-β and the related activin have been shown to induce the appearance of mesoderm from primitive neuroectoderm. TGF-β and related bone morphometric proteins can induce the differentiation of cartilage from primitive mesenchyme, and together with basic FGF and IGFs promote cartilage growth. Each class of growth factor is expressed within the epiphyseal growth plate where their autocrine/paracrine interactions regulate the rate of chondrocyte proliferation, matrix protein synthesis and terminal differentiation and mineralization. Basic FGF may prove useful in articular cartilage repair, while basic FGF, IGFs and TGF-β are among a number of growth factors and cytokines that have been implicated in cartilage disease.     

Molecular signaling in bone fracture healing and distraction osteogenesis

The process of fracture healing has been described in detail in many histological studies. Recent work has focused on the mechanisms by which growth and differentiation factors regulate the fracture healing process. Rapid progress in skeletal cellular and molecular biology has led to the identification of many signaling molecules associated with the formation of skeletal tissues, including members of the transforming growth factor-beta (TGF-beta) superfamily and the insulin-like growth factor (IGF) family. Increasing evidence indicates that they are critical regulators of cellular proliferation, differentiation, extracellular matrix biosynthesis and mineralization. Limb lengthening procedure (distraction osteogenesis) is a relevant model to investigate the in vivo correlation between mechanical stimulation and biological responses as the callus is stretched by a proper rate and rhythm of mechanical strain. This model also provides additional insights into the molecular and cellular events during bone fracture repair. TGF-beta 1 was significantly increased in both the distracted callus and the fracture callus. The increased level of TGF-beta 1, together with a low concentration of calcium and an enhanced level of collagen synthesis, was maintained in the distracted callus as long as mechanical strain was applied. Less mineralization is also associated with a low level of osteocalcin production. These observations provide further insights into the molecular basis for the cellular events during distraction osteogenesis.     

Chondrocyte BMP2 signaling plays an essential role in bone fracture healing

The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.     

Impairment of bone healing by insulin receptor substrate-1 deficiency

Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1(-/-) mice). Among 15 IRS-1(-/-) mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1(-/-) fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1(-/-) chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1(-/-) culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.     

Fibroblast growth factor (FGF) 18 signals through FGF receptor 3 to promote chondrogenesis

Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.     

Effects of diacerein on biosynthesis activities of chondrocytes in culture

The maintenance of articular cartilage integrity requires a balance between anabolic and catabolic processes which are under the control of chondrocytes. These cells are living in an anaerobic environment and normally do not divide. They are responsible for the continuous maintenance of the cartilage extracellular matrix (ECM). Although multiple factors are involved in the dynamic homeostasis of cartilage, increases in cytokines such as interleukin-1 (IL-1) are associated with a decrease in synthesis and an increase in degradation of the proteoglycans and collagens. Conversely, growth factors such as transforming growth factor-beta (TGF-beta) stimulate chondrocyte synthesis of collagens and proteoglycans, and reduce the activity of IL-1 stimulated metalloproteases, thus opposing the inhibitory and catabolic effects of IL-1. By its capability to reduce IL-1 effects and to stimulate TGF-beta expression in cultured articular chondrocytes, diacerein could favour anabolic processes in the OA cartilage and, hence may contribute to delay the progression of the disease.     

Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.