Mechanotransduction of bovine articular cartilage superficial zone protein by transforming growth factor β signaling

Objective

Mechanical signals are key determinants in tissue morphogenesis, maintenance, and restoration strategies in regenerative medicine, although molecular mechanisms of mechanotransduction remain to be elucidated. This study was undertaken to investigate the mechanotransduction process of expression of superficial zone protein (SZP), a critical joint lubricant.

Methods

Regional expression of SZP was first quantified in cartilage obtained from the femoral condyles of immature bovines, using immunoblotting, and visualized by immunohistochemistry. Contact pressure mapping in whole joints was accomplished using pressure‐sensitive film and a load application system for joint testing. Friction measurements on cartilage plugs were acquired under boundary lubrication conditions using a pin‐on‐disk tribometer modified for reciprocating sliding. Direct mechanical stimulation by shear loading of articular cartilage explants was performed with and without inhibition of transforming growth factor β (TGFβ) signaling, and SZP content in media was quantified by enzyme‐linked immunosorbent assay.

Results

An unexpected pattern of SZP localization in knee cartilage was initially identified, with anterior regions exhibiting high levels of SZP expression. Regional SZP patterns were regulated by mechanical signals and correlated with tribological behavior. Direct relationships were demonstrated between high levels of SZP expression, maximum contact pressures, and low friction coefficients. Levels of SZP expression and accumulation were increased by applying shear stress, depending on location within the knee, and were decreased to control levels with the use of a specific inhibitor of TGFβ receptor type I kinase and subsequent phospho‐Smad2/3 activity.

Conclusion

These findings indicate a new role for TGFβ signaling in the mechanism of cellular mechanotransduction that is especially significant for joint lubrication.

     

Articular cartilage repair by gene therapy using growth factor–producing mesenchymal cells

Objective

To investigate the repair of partial‐thickness lesions in rat articular cartilage by combining cell transplantation with transfer of growth factor complementary DNA (cDNA).

Methods

Mesenchymal cells isolated from rib perichondrium were infected ex vivo with adenoviral vectors carrying bone morphogenetic protein 2 (BMP‐2) or insulin‐like growth factor 1 (IGF‐1) cDNA. The cells were suspended in fibrin glue and applied to mechanically induced partial‐thickness cartilage lesions in the patellar groove of the rat femur. The filling of the defects was quantified and the quality and integration of the newly formed tissue were assessed by histochemical and immunohistochemical methods. Uninfected cells or cells infected with a LacZ reporter gene vector served as controls.

Results

Transplanted cells were able to attach to the wounded articular cartilage and were not displaced from the lesions by joint movement. Cells infected with both adenoviral vectors AdBMP‐2 and AdIGF‐1 produced repair cartilage of hyaline morphology containing a type II collagen–positive but type I collagen–negative proteoglycan‐rich matrix that restored the articular surface in most lesions. Uninfected cells either failed to fill up the defects or formed fibrous tissue mainly composed of type I collagen. Excessive cells were partially dislocated to the joint margins, leading to osteophyte formation there if AdBMP‐2–infected cells were used. These adverse effects, however, were not seen with AdIGF‐1–infected cells.

Conclusion

Stimulation of perichondrium‐derived mesenchymal cells by transfer of growth factor cDNA in a partial‐thickness defect model allows for satisfactory cartilage restoration by a repair tissue comparable with hyaline articular cartilage.

     

Transforming growth factor β–transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis

Objective

Bone marrow–derived mesenchymal stem cells (MSCs) can prevent various autoimmune diseases. We examined the therapeutic potential of transforming growth factor β (TGFβ)–transduced MSCs in experimental autoimmune arthritis, using an accepted animal model of collagen‐induced arthritis (CIA).

Methods

DBA/1J mice with CIA were treated with syngeneic TGFβ‐induced MSCs, whereas control mice received either vehicle or MSCs alone. Arthritis severity was assessed by clinical and histologic scoring. TGFβ‐transduced MSCs were tested for their immunosuppressive ability and differential regulation in mice with CIA. T cell responses to type II collagen were evaluated by determining proliferative capacity and cytokine levels. The effects of TGFβ‐transduced MSCs on osteoclast formation were analyzed in vitro and in vivo.

Results

Systemic infusion of syngeneic TGFβ‐transduced MSCs prevented arthritis development and reduced bone erosion and cartilage destruction. Treatment with TGFβ‐transduced MSCs potently suppressed type II collagen–specific T cell proliferation and down‐regulated proinflammatory cytokine production. These therapeutic effects were associated with an increase in type II collagen–specific CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the peritoneal cavity and spleen. Furthermore, TGFβ‐transduced MSCs inhibited osteoclast differentiation.

Conclusion

TGFβ‐transduced MSCs suppressed the development of autoimmune arthritis and joint inflammation. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell–mediated immunity using gene‐modified MSCs may be a gateway for new therapeutic approaches to clinical rheumatoid arthritis.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin‐1β and with chondrocyte resistance to insulin‐like growth factor 1

Objective

To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin‐1β (IL‐1β) and the responsiveness of isolated chondrocytes to stimulation with insulin‐like growth factor 1 (IGF‐1).

Methods

The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age‐related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0–7 for the presence of OA‐like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL‐1β. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF‐1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA‐related joint replacement surgery were also immunostained for nitrotyrosine and IL‐1β.

Results

The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL‐1β also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL‐1β was absent, suggesting that some age‐related oxidative damage is independent of IL‐1β. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL‐1β, the response to stimulation with IGF‐1 was significantly reduced. In some samples from older adult monkeys, IGF‐1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL‐1β.

Conclusion

Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF‐1 stimulation, but it is unlikely to be the sole cause of IGF‐1 resistance in these chondrocytes.

     

Synthesis of insulin‐like growth factor binding protein 3 in vitro in human articular cartilage cultures

Objective

To quantify the rate of synthesis of insulin‐like growth factor binding protein 3 (IGFBP‐3) and insulin‐like growth factor 1 (IGF‐1) by in vitro cultures of normal and osteoarthritic (OA) human articular cartilage.

Methods

Levels of IGF‐1 and IGFBP‐3 in media from in vitro cultures of human cartilage were determined by radioimmunoassay (RIA). IGFBPs were characterized by immunoblots and ligand blots. Ultrafiltration and RIA analysis of synovial fluid (SF) samples and washings of cartilage samples ex vivo were used to calculate partition coefficients and to estimate the amount of IGF‐1 and IGFBP‐3 in cartilage in vivo.

Results

OA cartilage synthesized 150 ng of IGFBP‐3 per gm of cartilage per day, compared with 50 ng synthesized by normal cartilage. The surface zone of normal cartilage produced more IGFBP‐3 than did the deep zone. Immunoblots and ligand blots confirmed the presence of IGFBP‐3. IGFBP‐3 synthesis was stimulated by exogenous IGF‐1. No freshly synthesized IGF‐1 was detected. The quantities of IGF‐1 and IGFBP‐3 present ex vivo were 11.3 and 78.7 ng/gm of cartilage in normal cartilage and 21.6 and 225.4 ng/gm in OA cartilage.

Conclusion

The results show that while IGFBP‐3 is synthesized in explant cultures, IGF‐1 is not. The rate of IGFBP‐3 synthesis is 3 times higher in OA than in normal cartilage. Both IGFBP‐3 and IGF‐1 penetrate into cartilage from SF in vivo. We estimate that the quantities of IGFBP‐3 produced in culture by human cartilage are small compared with the amount supplied in the form of “small complexes” from the circulation. The high value of the partition coefficient of IGFBP‐3 implies binding to the matrix.

     

Blocking vascular endothelial growth factor with soluble Flt‐1 improves the chondrogenic potential of mouse skeletal muscle–derived stem cells

Objective

To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on chondrogenesis, using muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle.

Methods

The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real‐time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP‐4) were coimplanted with MDSCs transduced to express either VEGF or sFlt‐1 (a VEGF antagonist) to provide a gain‐ and loss‐of‐function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups.

Results

Hyaline‐like cartilage matrix production was observed in both VEGF‐treated and VEGF‐blocked (sFlt‐1–treated) pellet cultures, but quantitative PCR revealed that sFlt‐1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP‐4 and transforming growth factor β3 (TGFβ3). In vivo testing of articular cartilage repair showed that VEGF‐transduced MDSCs caused an arthritic change in the knee joint, and sFlt‐1 improved the MDSC‐mediated repair of articular cartilage, compared with BMP‐4 alone.

Conclusion

Soluble Flt‐1 gene therapy improved the BMP‐4– and TGFβ3‐induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage.

     

Endothelin is a downstream mediator of profibrotic responses to transforming growth factor β in human lung fibroblasts

Objective

Fibrosis is excessive scarring caused by the accumulation and contraction of extracellular matrix proteins and is a common end pathway in many chronic diseases, including scleroderma (systemic sclerosis [SSc]). Indeed, pulmonary fibrosis is a major cause of death in SSc. Transforming growth factor β (TGFβ) induces endothelin 1 (ET‐1) in human lung fibroblasts by a Smad‐independent, JNK‐dependent mechanism. The goal of this study was to assess whether ET‐1 is a downstream mediator of the profibrotic effects of TGFβ in lung fibroblasts.

Methods

We used a specific endothelin receptor antagonist to determine whether ET‐1 is a downstream mediator of TGFβ responses in lung fibroblasts, using microarray technology, real‐time polymerase chain reaction, and Western blot analyses.

Results

The ability of TGFβ to induce the expression of a cohort of profibrotic genes, including type I collagen, fibronectin, and CCN2, and to contract a collagen gel matrix, depends on ET‐1.

Conclusion

ET‐1 contributes to the ability of TGFβ to promote a profibrotic phenotype in human lung fibroblasts, consistent with the notion that endothelin receptor antagonism may be beneficial in controlling fibrogenic responses in lung fibroblasts.

     

Chondrogenic differentiation of bovine synovium: Bone morphogenetic proteins 2 and 7 and transforming growth factor β1 induce the formation of different types of cartilaginous tissue

Objective

To compare the potential of bone morphogenetic proteins 2 and 7 (BMP‐2 and BMP‐7) and transforming growth factor β1 (TGFβ1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed.

Methods

Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP‐2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP‐2, BMP‐7, and TGFβ1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level.

Results

BMP‐2 induced the chondrogenic differentiation of synovial explants in a dose‐ and time‐dependent manner under serum‐ and dexamethasone‐free conditions. The expression levels of cartilage‐related genes increased in a time‐dependent manner. BMP‐7 was more potent than BMP‐2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFβ1 differed in terms of both cell phenotype and gene expression profiles.

Conclusion

The 3 tested members of the TGFβ superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

     

Intraarticular injection of heparin‐binding insulin‐like growth factor 1 sustains delivery of insulin‐like growth factor 1 to cartilage through binding to chondroitin sulfate

Objective

Insulin‐like growth factor 1 (IGF‐1) stimulates cartilage repair but is not a practical therapy due to its short half‐life. We have previously modified IGF‐1 by adding a heparin‐binding domain and have shown that this fusion protein (HB‐IGF‐1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB‐IGF‐1 is retained in cartilage and to test whether HB‐IGF‐1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants.

Methods

Retention of HB‐IGF‐1 and IGF‐1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB‐IGF‐1 and IGF‐1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme‐linked immunosorbent assay.

Results

In cartilage explants, chondroitinase treatment decreased binding of HB‐IGF‐1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB‐IGF‐1 retention on cell monolayers. Binding assays showed that HB‐IGF‐1 bound both CS and HS, whereas IGF‐1 did not bind either. After intraarticular injection in rat knees, HB‐IGF‐1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS‐rich cartilage. Finally, HB‐IGF‐1 was retained in human cartilage explants but IGF‐1 was not.

Conclusion

Our findings indicate that after intraarticular injection in rats, HB‐IGF‐1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin‐binding domains may be a new strategy for sustained and specific local delivery to cartilage.

     

Oncostatin M in combination with tumor necrosis factor α induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo

Objective

To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems.

Methods

The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα.

Results

The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP‐1), MMP‐3, and MMP‐13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP‐1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP‐1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone.

Conclusion

This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

     

Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor β/bone morphogenetic protein inhibitors during experimental osteoarthritis

Objective

Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor β (TGFβ) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFβ and BMP during papain‐induced OA‐like changes in mice.

Methods

We used adenoviral overexpression of TGFβ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan–specific TGFβ antagonist called murine latency‐associated peptide 1 (mLAP‐1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFβ/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.

Results

Intraarticular injection of papain caused increased protein expression of several TGFβ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP‐1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFβ inhibitor mLAP‐1 increased articular cartilage PG loss.

Conclusion

Our results indicate a pivotal role of endogenous TGFβ in the development of osteophytes and synovial thickening, implicating endogenous TGFβ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFβ signifies the protective role of TGFβ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.

     

Involvement of αvβ5 integrin–mediated activation of latent transforming growth factor β1 in autocrine transforming growth factor β signaling in systemic sclerosis fibroblasts

Objective

To confirm the involvement of αvβ5 in the self‐activation system in systemic sclerosis (SSc) fibroblasts.

Methods

Levels of αvβ5 expression were analyzed by immunoprecipitation. The promoter activity of the human α2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor β (TGFβ) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.

Results

Levels of αvβ5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti‐αvβ5 antibody or β5 antisense oligonucleotide significantly reduced human α2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFβ1 antisense oligonucleotide, the exogenous latent TGFβ1 stimulation significantly increased human α2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti‐αvβ5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with β5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti‐αvβ5 antibody. Anti‐αvβ5 antibody reversed the myofibroblastic features of SSc fibroblasts.

Conclusion

Up‐regulated expression of αvβ5 contributes to the establishment of autocrine TGFβ signaling in SSc fibroblasts through activation of endogenous latent TGFβ1.

     

Lack of evidence of stimulatory autoantibodies to platelet‐derived growth factor receptor in patients with systemic sclerosis

Objective

Systemic sclerosis (SSc) is a severe connective tissue disease of unknown etiology, characterized by fibrosis of the skin and multiple internal organs. Recent findings suggested that the disease is driven by stimulatory autoantibodies to platelet‐derived growth factor receptor (PDGFR), which stimulate the production of reactive oxygen species (ROS) and collagen by fibroblasts. These results opened novel avenues of research into the diagnosis and treatment of SSc. The present study was undertaken to confirm the presence of anti‐PDGFR antibodies in patients with SSc.

Methods

Immunoglobulins from 37 patients with SSc were purified by protein A/G chromatography. PDGFR activation was tested using 4 different sensitive bioassays, i.e., cell proliferation, ROS production, signal transduction, and receptor phosphorylation; the latter was also tested in a separate population of 7 patients with SSc from a different research center.

Results

Purified IgG samples from patients with SSc were positive when tested for antinuclear autoantibodies, but did not specifically activate PDGFRα or PDGFRβ in any of the tests. Cell stimulation with PDGF itself consistently produced a strong signal.

Conclusion

The present results raise questions regarding the existence of agonistic autoantibodies to PDGFR in SSc.

     

Basic fibroblast growth factor inhibits the anabolic activity of insulin‐like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

Objective

To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin‐like growth factor 1 (IGF‐1) and osteogenic protein 1 (OP‐1).

Methods

Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum‐free medium with or without IGF‐1 (100 ng/ml), OP‐1 (100 ng/ml), or bFGF (0–100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants.

Results

Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF‐1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF‐1 and OP‐1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF‐1 or OP‐1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF‐1 and OP‐1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures.

Conclusion

The anabolic activity of IGF‐1 and OP‐1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.