bFGF influences human articular chondrocyte differentiation

BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro in monolayer. bFGF-dependent cell proliferation, production of collagen type II and aggrecan were monitored 10 days after isolation. Furthermore, effect of bFGF on cell cycle, cell morphology, and mRNA expression of integrins and chondrogenic markers determined by real time PCR were analyzed. RESULTS: bFGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained unchanged. Supplementation of cell culture with bFGF reduced collagen type II mRNA by 49%, but increased expression of the integrin alpha(2) by 70%. bFGF did not significantly regulate the integrins alpha(1), alpha(5), alpha(10), alpha(v) and type I collagen. bFGF reduced the amount of collagen type II by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential role of bFGF as an endogenous chondrocyte mediator that can enhance cell amplification and regulate cell differentiation.     

FAK mediates signal crosstalk between type II collagen and TGF-beta 1 cascades in chondrocytic cells

The purpose of this study was to evaluate the mechanism of crosstalk between the type II collagen and TGF-β1 signaling pathways in chondrocytic cells. Articular chondrocytes, isolated from porcine knee cartilage, and the SW1353 cell line were cultured on either type II collagen-coated or -uncoated plates in the presence or absence of TGF-β1. Expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 in articular chondrocytes and the SW1353 cell line was analyzed by immunoblotting. Cell proliferation rates and glycosaminoglycan (GAG) content was determined after treatment with type II collagen or/and TGF-β1. For inhibition study, human FAK-specific RNA small interference (siFAK) in SW1353 cell line was performed. In this study, expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 were synergistically increased by co-treatment with type II collagen and TGF-β1 in articular chondrocytes. The proliferation of porcine articular chondrocytes and GAG secretion in SW1353 cells were synergistically increased by co-stimulation with type II collagen and TGF-β1. Synergistically increased expression and nuclear translocation of pSMAD 2 and pSMAD 3 and GAG secretion of SW1353 cells were significantly inhibited by siFAK transfection. Therefore, we suggest that FAK-SMAD 2/3 mediates signal crosstalk between type II collagen and TGF-β1 and regulates GAG secretion in chondrocytic cells.     

Effects of Strontium on Collagen Content and Expression of Related Genes in Rat Chondrocytes Cultured In Vitro

Strontium stimulates cartilage matrix formation in vitro. However, the mechanisms governing these effects have not yet been extensively reported. In this study, chondrocytes were isolated from rat articular cartilage by enzymatic digestion and cultured for 24–72 h with 1–5 mM strontium. We investigated the effects of different concentrations of strontium on collagen content, type II collagen, insulin-like growth factor (IGF-1) and matrix metalloproteinase (MMP)-13 expression in rat cultured articular chondrocytes in vitro. The collagen content of the chondrocytes, determined as hydroxyproline, was measured by a colorimetry method. Type II collagen, IGF-1, and MMP-13 mRNA abundance and protein expression levels were determined by real-time polymerase chain reaction (real-time PCR) and western blot, respectively. The results showed that collagen content from the chondrocytes extracellular matrix increased with increasing strontium concentration. Moreover, 3 and 5 mM strontium strongly stimulated protein expression and mRNA levels of type II collagen and IGF-1. Conversely, MMP-13 expression in chondrocytes decreased dose-dependently with increasing strontium concentration. These results should provide insight into the ability of strontium to promote chondrocyte extracellular matrix synthesis. Strontium could promote collagen synthesis and suppress collagen degradation via the repression of MMP-13 expression.     

Endocytosis of different members of the small chondroitin/dermatan sulfate proteoglycan family.

The family of small interstitial chondroitin/dermatan sulfate proteoglycans consists of at least three different molecular species: biglycan (proteoglycan I), decorin (proteoglycan II), and proteoglycan-100, which has a glycosylated core protein of about 100 kDa. The core protein of decorin has been shown to be responsible for receptor-mediated endocytosis of this proteoglycan species by a variety of mesenchymal cells. It is now demonstrated that skin fibroblasts and articular chondrocytes endocytose biglycan with an efficiency similar to that of decorin. Uptake of biglycan is also mediated by its core protein and can be inhibited by decorin in a partially competitive manner. In human fibroblasts, endosomal proteins of 51 and 26 kDa, which are known to bind decorin core protein, also interact with biglycan. This interaction can be inhibited by decorin. Bovine articular chondrocytes contained binding proteins of 48 and 25 kDa. Proteoglycan-100 can be distinguished from biglycan and decorin by its low clearance rate, which however, exceeds the rate of fluid phase endocytosis.     

Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis

Osteoarthritis (OA), the commonest form of arthritis and a major cause of morbidity, is characterized by progressive degeneration of the articular cartilage. Along with increased production and activation of degradative enzymes, altered synthesis of cartilage matrix molecules and growth factors by resident chondrocytes is believed to play a central role in this pathological process. We used an ovine meniscectomy model of OA to evaluate changes in chondrocyte expression of types I, II and III collagen; aggrecan; the small leucine-rich proteoglycans (SLRPs) biglycan, decorin, lumican and fibromodulin; transforming growth factor-β; and connective tissue growth factor. Changes were evaluated separately in the medial and lateral tibial plateaux, and were confirmed for selected molecules using immunohistochemistry and Western blotting. Significant changes in mRNA levels were confined to the lateral compartment, where active cartilage degeneration was observed. In this region there was significant upregulation in expession of types I, II and III collagen, aggrecan, biglycan and lumican, concomitant with downregulation of decorin and connective tissue growth factor. The increases in type I and III collagen mRNA were accompanied by increased immunostaining for these proteins in cartilage. The upregulated lumican expression in degenerative cartilage was associated with increased lumican core protein deficient in keratan sulphate side-chains. Furthermore, there was evidence of significant fragmentation of SLRPs in both normal and arthritic tissue, with specific catabolites of biglycan and fibromodulin identified only in the cartilage from meniscectomized joints. This study highlights the focal nature of the degenerative changes that occur in OA cartilage and suggests that altered synthesis and proteolysis of SLRPs may play an important role in cartilage destruction in arthritis.     

Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

Modulation of sulfated proteoglycan synthesis and collagen gene expression by chondrocytes grown in the presence of bFGF alone or combined with IGF1

Prepubertal rabbit epiphyseal chondrocytes were grown in high density primary culture for 3 d. They were then incubated for 3 additional d in serum-free culture medium to which bFGF (1-50 ng/ml) was added. During the last 24 h incubation period, either IGF1 (1-80 ng/ml) or Insulin (1-5 micrograms/ml) was added to the culture medium. Chondrocyte DNA was significantly augmented with the increasing concentration of bFGF used, thus confirming its mitogenic effect on chondrocytes. On the other hand, bFGF was also shown to modulate the phenotypic expression of the chondrocytes. The 35S-sulfate incorporation into newly synthesized proteoglycans by the cultured cells decreased in a dose-dependent manner with bFGF concentration used. In addition, chondrocyte collagen gene expression was also shown to be modulated by bFGF. Total RNA extracted from the cultured cells was analyzed by dot blot and Northern blot with cDNA probes encoding for alpha 1 II and alpha 1 I procollagen chains. A significant lower level of type II collagen mRNA, the marker of chondrocytic phenotype, was observed when cells were grown in the presence of bFGF while the level of type I mRNA remained unchanged. When IGF1 or a high concentration of insulin was added to the cells during the last 24 h of incubation with bFGF, sulfated proteoglycan synthesis, as well as collagen type II mRNA level, were significantly stimulated when compared with chondrocytes incubated with bFGF alone. In conclusion, in the present experimental conditions, bFGF appears to be a growth promoting agent for chondrocytes in vitro with dedifferentiating action on chondrocyte phenotype. IGF1 or insulin used at a high concentration can prevent the dedifferentiating effect of bFGF without inhibiting its stimulating effect on chondrocyte DNA synthesis.     

Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)     

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10⁵ cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.     

Light and electron microscopical immunohistochemical localization of the small proteoglycan core proteins decorin and biglycan in human knee joint cartilage

Summary The distribution of decorin and biglycan was investigated at the light and electron microscopical level in adult human articular cartilage. In general, the amount of decorin and biglycan was found to decrease with the depth of the layer of the cartilage. Decorin was found in the interterritorial matrix where most of the collagen is located. This fits in well with the assumption that decorin may modulate collagen metabolism. Biglycan was found next to the chondrocytes in the pericellular matrix and is assumed to be responsible for cellular activities. At the ultrastructural level, decorin was localized in the interterritorial matrix and in vesicles in chondrocytes. Biglycan was found, usually though not exclusively in the pericellular matrix. Both small proteoglycans were detected close to and on the collagen fibres and also associated with the more globular structures of the matrix between the fibrils. A double-staining approach revealed that the two molecules could be located along the same collagen fibril. However, staining for biglycan and decorin was not observed simultaneously within the same region of the fibre.     

Fibrils of different collagen types containing immobilised proteoglycans (PGs) as coatings: characterisation and influence on osteoblast behaviour

Collagen, the main organic component of bone, is used as a coating on titanium implants and as a scaffold material in bone tissue engineering. Surface modifications of titanium which promote osteoblast adhesion, proliferation and synthesis of collagen by osteoblasts are desirable. One biomimetic approach is the coating of titanium with collagen in fibrillar form. Other organic components of bone may be bound to fibrils and exert additional effects. In this study, the collagen types I-III were compared regarding their ability to bind the proteoglycans decorin and biglycan, which are found in bone. More collagen was bound to collagen II fibrils than to those of types I and III. Therefore, titanium surfaces were coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the effect of the proteoglycans on human primary osteoblast behaviour. In addition, the growth factor TGF-beta1 was adsorbed onto surfaces coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the influence of decorin and biglycan on the effect of TGF-beta1 on osteoblasts. Fibril-bound biglycan and decorin influence primary osteoblast behaviour by themselves. The presence of substrate-bound biglycan or decorin influences the effect of TGF-beta1. These results may be important when designing collagen-based coatings or scaffolds for tissue engineering, including those loaded with growth factors.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

New aspects of the pathogenesis of osteoarthritis: the role of fibroblast-like chondrocytes in late stages of the disease

It is thought that the general increase in life expectancy will make osteoarthritis the fourth leading cause of disability by the year 2020. Even though the pathogenesis of idiopathic osteoarthritis has not been fully elucidated, the main features of the disease process are the altered interactions between the chondrocytes and their surrounding extracellular matrix. In the course of these disturbances, three types of chondrocytes are typically present in the pathologically altered extracellular matrix of the articular cartilage: healthy chondrocytes which are continually undergoing degeneration, degenerated cells which are continually being degraded and finally fibroblast-like chondrocytes which seem not to be influenced by this process and, therefore, are found in ever-increasing numbers. These fibroblast-like chondrocytes take part in tissue regeneration even in advanced stages of osteoarthritis, but only in as much as they form fibrocartilaginous or scar tissue, since, as we were able to show, they mainly synthesize collagen type I and not collagen type II, typical for healthy cartilage. However, we were further able to show that fibroblast-like chondrocytes also produce increasing amounts of the proteoglycans decorin and biglycan which physiologically are involved in the formation of collagen type II, as well as perlecan. These multifunctional fibroblast-like chondrocytes could present an ideal therapeutic starting point if they could be modified to synthesize the collagen type II typical for cartilage and to, thereby, contribute to reversing the damage of the joint cartilage that has occurred by the late stages of osteoarthritis.     

In vitro insulin-like growth factor I interaction with cartilage cells derived from postnatal animals

This paper reports data on the in vitro effects of insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) on the phenotypic expression of epiphyseal chondrocytes grown in serum-free (SF) culture medium. bFGF mostly stimulates chondrocyte DNA and inhibits sulfated proteoglycan synthesis and type II collagen mRNA. On the contrary, IGF-I is poorly mitogenic but strongly stimulates protein synthesis and type II collagen mRNA. In addition, IGF-I prevents the expression of type I collagen gene. Lastly, chondrocytes cultured in SF medium are able to locally produce IGF-I peptides. In conclusion, IGF-I and bFGF have opposite effects on the phenotypic expression of chondrocytes in vitro: bFGF is mostly mitogenic and IGF-I appears to be a differentiating factor.     

Dynamic compression of rabbit adipose-derived stem cells transfected with insulin-like growth factor 1 in chitosan/gelatin scaffolds induces chondrogenesis and matrix biosynthesis

Articular cartilage is routinely subjected to mechanical forces and growth factors. Adipose-derived stem cells (ASCs) are multi-potent adult stem cells and capable of chondrogenesis. In the present study, we investigated the comparative and interactive effects of dynamic compression and insulin-like growth factor-I (IGF-I) on the chondrogenesis of rabbit ASCs in chitosan/gelatin scaffolds. Rabbit ASCs with or without a plasmid overexpressing of human IGF-1 were cultured in chitosan/gelatin scaffolds for 2 days, then subjected to cyclic compression with 5% strain and 1 Hz for 4 h per day for seven consecutive days. Dynamic compression induced chondrogenesis of rabbit ASCs by activating calcium signaling pathways and up-regulating the expression of Sox-9. Dynamic compression plus IGF-1 overexpression up-regulated expression of chondrocyte-specific extracellular matrix genes including type II collagen, Sox-9, and aggrecan with no effect on type X collagen expression. Furthermore, dynamic compression and IGF-1 expression promoted cellular proliferation and the deposition of proteoglycan and collagen. Intracellular calcium ion concentration and peak currents of Ca(2+) ion channels were consistent with chondrocytes. The tissue-engineered cartilage from this process had excellent mechanical properties. When applied together, the effects achieved by the two stimuli (dynamic compression and IGF-1) were greater than those achieved by either stimulus alone. Our results suggest that dynamic compression combined with IGF-1 overexpression might benefit articular cartilage tissue engineering in cartilage regeneration.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.     

Regulation of type II collagen expression by cyclin-dependent kinase 6, cyclin D1, and p21 in articular chondrocytes

This study examined whether cell cycle regulatory proteins, such as cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors, regulate type II collagen expression and mediate interlukin-1 (IL-1beta)-induced suppression of type II collagen expression in articular chondrocytes. IL-1beta inhibited type II collagen expression, but activated CDK6. Ectopic expression of CDK2 did not alter type II collagen expression. However, overexpression of CDK6 inhibited type II collagen expression, whereas inhibition of CDK6 activity blocked IL-1beta-induced suppression of type II collagen expression. IL-1beta upregulated the expression of cyclin D1, which is known to activate CDK6. In turn, overexpression of cyclin D1 suppressed type II collagen expression. In contrast to cyclin D1, IL-1beta triggered down-regulation of the CDK inhibitor, p21. Overexpression of p21 blocked IL-1beta- or CDK6-induced suppression of type II collagen expression. Our results collectively indicate that CDK6/cyclin D1/p21 complex regulates type II collagen expression in articular chondrocytes.     

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.