Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing

The transplantation of chondrocytes has shown promise for augmenting the repair of defects in articular cartilage. This in vitro study examined the efficiency of the transplantation of bovine chondrocytes onto articular cartilage disks and the ability of the transplanted chondrocytes to subsequently synthesize and deposit proteoglycan. The radiolabeling of chondrocyte cultures with [3H]thymidine, followed by 4 days of chase incubation, resulted in the incorporation of 98% of the radiolabel into DNA (as assessed by susceptibility to DNase). At the end of the culture period, the [3H]DNA was stable, with a half-life of radioactivity loss into the medium of 73 days. With use of radiolabeled chondrocytes for quantitation, the efficiency of transplantation onto a cartilage substrate was 93 +/- 4% for seeding densities of as much as 650,000 cells per cm2 and a seeding duration of 1 hour. These findings were confirmed both by tracking cells stained with 5-chlormethylfluorescein diacetate and by quantitating DNA. During the 16 hours after seeding onto a cartilage substrate (in which the endogenous cells had been lysed by lyophilization), the transplanted cells synthesized sulfated proteoglycan in direct proportion to the number of cells seeded. Most (83%) of the newly synthesized proteoglycan was released into the medium rather than retained within the layer of transplanted cells and the recipient cartilage substrate. Comparative studies with lyophilized-rehydrated or live cartilage as the recipient substrate indicated a similar efficiency of chondrocyte seeding and proteoglycan synthesis by the seeded chondrocytes. The transplanted cells retained the chondrocyte phenotype, as judged by a high proportion of the [35S]macromolecules being in the form of aggrecan that was capable of aggregating with hyaluronan and link protein, as well as by immunostaining within and around the transplanted cells for type-II, but not type-I, collagen. These results indicate that the number of chondrocytes transplanted onto a cut cartilage surface greatly affects the level of matrix synthesis; this in turn may affect repair.     

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.     

Cartilage cell transplantation. Experimental principles and clinical applications]

Transplantation of autologous chondrocytes proliferated in vitro to treat cartilage defects is discussed controversially. Cartilage has only a very limited capacity to regenerate. The repair tissue is of minor biomechanical stability and therefore induces degenerative arthritis of joints in the long term. Facial cartilage defects may lead to aesthetic or functional problems. Different biological and synthetic substitutes were used to treat cartilage defects in orthopaedic and facial plastic surgery. Biological tissue for transplantation is not abundant and synthetic materials may induce foreign body reactions. Amplification of autologous cells in vitro to produce a tissue similar to the impaired tissue seems tempting. It is transplantable into the defect and is tough to restore the defective surface completely. This is the intention of numerous scientific investigations concerning chondrocyte application to cartilage defects. Transplantation of isolated chondrocytes is currently used in clinical trials also. The possibilities and limitations of this technique are discussed including the fundamental principles and our own experimental investigations. A proof for the reproduction of a tissue similar to native cartilage with its typical long term mechanical properties is still missing. Further laboratory and clinical studies should be conducted before the technique is propagated in patient care.     

Chondrogenic potential of skeletal cell populations: selective growth of chondrocytes and their morphogenesis and development in vitro

Most vertebrate embryonic and post-embryonic skeletal tissue formation occurs through the endochondral process in which cartilage serves a transitory role as the anlage for the bone structure. The differentiation of chondrocytes during this process in vivo is characterized by progressive morphological changes associated with the hypertrophy of these cells and is defined by biochemical changes that result in the mineralization of the extracellular matrix. The mechanisms, which, like those in vivo, promote both chondrogenesis in presumptive skeletal cell populations and endochondral progression of chondrogenic cells, may be examined in vitro. The work presented here describes mechanisms by which cells within presumptive skeletal cell populations become restricted to a chondrogenic lineage as studied within cell populations derived from 12-day-old chicken embryo calvarial tissue. It is found that a major factor associated with selection of chondrogenic cells is the elimination of growth within serum-containing medium. Chondrogenesis within these cell populations appears to be the result of permissive conditions which select for chondrogenic proliferation over osteogenic cell proliferation. Data suggest that chondrocyte cultures produce autocrine factors that promote their own survival or proliferation. The conditions for promoting cell growth, hypertrophy, and extracellular matrix mineralization of embryonic chicken chondrocytes in vitro include ascorbic acid supplementation and the presence of an organic phosphate source. The differentiation of hypertrophic chondrocytes in vitro is associated with a 10-15-fold increase in alkaline phosphatase enzyme activity and deposition of mineral within the extracellular matrix. Temporal studies of the biochemical changes coincident with development of hypertrophy in vitro demonstrate that proteoglycan synthesis decreases 4-fold whereas type X collagen synthesis increases 10-fold within the same period. Ultrastructural examination reveals cellular and extracellular morphology similar to that of hypertrophic cells in vivo with chondrocytes embedded in a well formed extracellular matrix of randomly distributed collagen fibrils and proteoglycan. Mineral deposition is seen in the interterritorial regions of the matrix between the cells and is apatitic in nature. These characteristics of chondrogenic growth and development are very similar in vivo and in vitro and they suggest that studies of chondrogenesis in vitro may provide a valuable model for the process in vivo.     

High-frequency oscillation and centroid frequency of diaphragm EMG during inspiratory loading

Exposure of progenitor cells with chondrogenic potential to recombinant human osteogenic protein-1 [rhOP-1, or bone morphogenetic protein-7 (BMP-7] may be of therapeutic interest in the regeneration of articular cartilage. Therefore, in this study, we examined the influence of rhOP-1 on cartilage formation by human perichondrium tissue containing progenitor cells with chondrogenic potential in vitro. Fragments of outer ear perichondrium tissue were embedded in clotting autologous blood to which rhOP-1 had been added or not (controls), and the resulting explant was cultured for 3 weeks without further addition of rhOP-1. Cartilage formation was monitored biochemically by measuring [³5;S]sulfate incorporation into proteoglycans and histologically by monitoring the presence of metachromatic matrix with cells in nests. The presence of rhOP-1 in the explant at the beginning of culture stimulated [³5;S]sulfate incorporation into proteoglycans in a dose-dependent manner after 3 weeks of culture. Maximal stimulation was reached at 40 microgram/ml. Histology revealed that explants treated with 20-200 microgram/ml rhOP-1, but not untreated control explants, contained areas of metachromatic-staining matrix with chondrocytes in cell nests. These results suggest that rhOP-1 stimulates differentiation of cartilage from perichondrium tissue. The direct actions of rhOP-1 on perichondrium cells to stimulate chondrocytic differentiation and production of cartilage matrix in vitro provide a cellular mechanism for the induction of cartilage formation by rhOP-1 in vivo. Thus, rhOP-1 may promote early steps in the cascade of events leading to cartilage formation. Therefore, rhOP-1 could be an interesting factor for regeneration of cartilage in articular cartilage defects.     

Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture

Chondrocytes propagated in monolayer culture proliferate and change into 'fibroblastoid'-like cells. This change is characterized by a shift in production of collagen type II to I and from high- to low-molecular-weight proteoglycans. When propagated in three-dimensional culture, chondrocytes have limited ability to divide but re-express their original characteristics. The goal of the present study was to determine whether a microcarrier suspension culture system would support chondrocyte proliferation and phenotype expression. Our experiments indicate that a collagen type I microcarrier (cellagen) best supported chondrocyte proliferation and phenotype expression. Cells in cellagen microcarriers multiplied at least twentyfold within 2 weeks and had doubling times of 2 to 3 d. Viable and metabolically active cells were retrieved with ease. The harvested chondrocytes had no detectable staining for collagen type I and stained intensely for collagen type II. Our studies demonstrate that the microcarrier suspension culture system supports growth and enhances expression of the 'chondrocytic' phenotype. Attachment to a constrained surface and the fluid shear forces on the microcarriers during suspension culture may have helped chondrocytes to reacquire their rounded shape and produce cartilage matrix components.     

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

Effect of cultured autologous chondrocytes on repair of chondral defects in a canine model

Articular cartilage has a limited capacity for repair. In recent clinical and animal experiments, investigators have attempted to elicit the repair of defects of articular cartilage by injecting cultured autologous chondrocytes under a periosteal flap (a layer of periosteum). The objective of the present study was to determine the effect of cultured autologous chondrocytes on healing in an adult canine model with use of histomorphometric methods to assess the degree of repair. A total of forty-four four-millimeter-diameter circular defects were created down to the zone of calcified cartilage in the articular cartilage of the trochlear groove of the distal part of the femur in fourteen dogs. The morphology and characteristics of the original defects were defined in an additional six freshly created defects in three other dogs. Some residual noncalcified articular cartilage, occupying approximately 2 per cent of the total cross-sectional area of the defect, was sometimes left in the defect. The procedure sometimes damaged the calcified cartilage, resulting in occasional microfractures or larger fractures, thinning of the zone of calcified cartilage, or, rarely, small localized penetrations into subchondral bone. The forty-four defects were divided into three treatment groups. In one group, cultured autologous chondrocytes were implanted under a periosteal flap. In the second group, the defect was covered with a periosteal flap but no autologous chondrocytes were implanted. In the third group (the control group), the defects were left empty. The defects were analyzed after twelve or eighteen months of healing. Histomorphometric measurements were made of the percentage of the total area of the defect that became filled with repair tissue, the types of tissue that filled the defect, and the integration of the repair tissue with the adjacent cartilage at the sides of the defects and with the calcified cartilage at the base of the defect. In histological sections made through the center of the defects in the three groups, the area of the defect that filled with new repair tissue ranged from a mean total value of 36 to 76 per cent, with 10 to 23 per cent of the total area consisting of hyaline cartilage. Integration of the repair tissue with the adjacent cartilage at the edges of the defect ranged from 16 to 32 per cent in the three groups. Bonding between the repair tissue and the calcified cartilage at the base of the defect ranged from 41 to 89 per cent. With the numbers available, we could detect no significant difference among the three groups with regard to any of the parameters used to assess the quality of the repair. In the two groups in which a periosteal flap was sutured to the articular cartilage surrounding the defect, the articular cartilage showed degenerative changes that appeared to be related to that suturing.     

Rabbit articular cartilage defects treated with autologous cultured chondrocytes

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.     

Parathyroid hormone-related proteins is abundant in osteoarthritic cartilage, and the parathyroid hormone-related protein 1-173 isoform is selectively induced by transforming growth factor beta in articular chondrocytes and suppresses generation of extracellular inorganic pyrophosphate

OBJECTIVE: Parathyroid hormone-related protein (PTHrP) is a major, locally expressed regulator of growth cartilage chondrocyte proliferation, differentiation, synthetic function, and mineralization. Because mechanisms that limit cartilage chondrocytes from maturing and mineralizing are diminished in osteoarthritis (OA), we studied PTHrP expression by articular chondrocytes. METHODS: PTHrP was studied in normal knee cartilage samples and cultured articular chondrocytes, and in cartilage specimens from knees with advanced OA, obtained at the time of joint replacement. RESULTS: PTHrP was more abundant in OA than in normal human knee articular cartilage. Both demonstrated PTH/PTHrP receptor expression. PTHrP 1-173, one of three alternatively spliced PTHrP isoforms, was exclusively expressed and induced by transforming growth factor beta in cultured chondrocytes. Chondrocytes mainly used the GC-rich P2 alternative promoter to express PTHrP messenger RNA. Inhibition by PTHrP 1-173, but not by PTHrP 1-146 or PTHrP 1-87, of inorganic pyrophosphate (PPi) elaboration suggested selective functional properties of the 1-173 isoform. Exposure to a neutralizing antibody to PTHrP increased PPi elaboration by articular chondrocytes. CONCLUSION: Increased expression of PTHrP, including the 1-173 isoform, has the potential to contribute to the pathologic differentiated functions of chondrocytes, including mineralization, in OA.     

Terminal differentiation of chondrocytes is arrested at distinct stages identified by their expression repertoire of marker genes

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.     

Electrocardiographically gated myocardial perfusion SPECT: technical principles and quality control considerations

BACKGROUND: Apoptosis in vivo has been identified in developing cartilage from embryonic chick sterna and avian and murine growth plates. To date, no evidence exists that chondrocytes in articular cartilage undergo apoptosis. METHODS: We examined the distribution of cells demonstrating fragmented DNA in the articular knee cartilage of C57BL/6 mice (aged 11, 18, 24, and 30 months) and Wistar rats (aged 6, 12, and 24 months) using a DNA end-labeling technique. RESULTS: Control experiments utilizing retinoic acid-induced apoptosis in a chondrocyte cell line, established that DNA end-labeling correlated with DNA ladder formation. In vivo, apoptotic cells were detected in articular cartilage tissue in both species examined. The percentage of apoptotic cells increased significantly (P < 0.05 with age) for all joint surfaces in both species. No significant difference was found between the medial and lateral or femoral and tibial joint surfaces of the knee. Apoptotic cells were observed in both the calcified and uncalcified regions of the articular cartilage of C57 mice. In the rat, only the calcified region of articular cartilage contained apoptotic cells. CONCLUSIONS: These results suggest that apoptosis plays a role in some aspect of maintenance, remodeling, or turnover of mature articular cartilage. In addition, the increase in apoptosis associated with aging could contribute to the greater risk for cartilage degeneration.     

Effect of a combined trenbolone acetate and estradiol implant on steady-state IGF-I mRNA concentrations in the liver of wethers and the longissimus muscle of steers

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. It is well established that the subchondral vascular system is pivotal in the regulation of this process. Cells of subchondral blood vessels act as a source of vascular invasion and, in addition, release factors influencing growth and differentiation of chondrocytes in the avascular growth plate. To elucidate the paracrine contribution of endothelial cells we studied the hypertrophic development of resting chondrocytes from the caudal third of chick embryo sterna in co-culture with endothelial cells. The design of the experiments prevented cell-to-cell contact but allowed paracrine communication between endothelial cells and chondrocytes. Under these conditions, chondrocytes rapidly became hypertrophied in vitro and expressed the stage-specific markers collagen X and alkaline phosphatase. This development also required signaling by thyroid hormone in synergy. Conditioned media could replace the endothelial cells, indicating that diffusible factors mediated this process. By contrast, smooth muscle cells, fibroblasts, or hypertrophic chondrocytes did not secrete this activity, suggesting that the factors were specific for endothelial cells. We conclude that endochondral ossification is under the control of a mutual communication between chondrocytes and endothelial cells. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.     

Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes

Under certain conditions chondrocytes form lattices with cartilage collagens, which may serve as cartilage implants. It is necessary to find the optimal conditions for culturing chondrocytes. Three different supports are compared: (a) plastic; (b) cartilage collagens; and (c) insoluble skin collagen solubilized under denaturing conditions (ISC-40). The effect of culture medium supplementation with the tripeptide (Gly-His-Lys)2.Cu.2H2O.2NaCl (GHK) on chondrocyte proliferation and synthetic activity is studied, with particular attention paid to collagen types I, II and III. The collagen supports stimulated chondrocyte proliferation, but on the ISC-40 support they started to dedifferentiate rather early. In the primary culture, chondrocytes on all three supports synthesized mainly collagen type II, and only small amounts of types I and III. In the first passage the synthesis of these two collagen types increased, relative to collagen type II, at least on the cartilage collagen support. Supplementation of culture medium with GHK stimulated chondrocyte proliferation in the primary structure mostly on the ISC-40 support. On the other two types of supports the stimulatory effect of GHK was expressed mostly in the first passages. The collagen synthetic rate was increased by GHK on both of the collagen supports; on the cartilage collagen support collagen type II was synthesized predominantly and on the ISC-40 support types I and III were mostly formed. It is suggested that supplementation of culture medium with GHK may be useful in the preparation of cartilage implants.     

A novel cartilage protein (CILP) present in the mid-zone of human articular cartilage increases with age

A novel, somewhat basic noncollagenous protein was purified from guanidine hydrochloride extracts of human articular cartilage using cesium chloride density gradient centrifugation, followed by ion-exchange chromatography at pH 5, and gel filtration on two serially coupled columns of Superose 6 and Superdex 200. The protein of 91.5 kDa contains a single polypeptide chain substituted with N-linked oligosaccharides. It appeared unique to cartilage as studied by enzyme-linked immunosorbent assay and immunoblots of various tissue extracts. Its concentration in articular cartilages showed some variability with age being lower in young individuals. It represents a chondrocyte product, since it is synthesized by articular chondrocytes in explant cultures. Interestingly, the distribution of the protein in the articular cartilage provides important information on the nature of chondrocytes at different compartments in the tissue. Thus, chondrocytes in the middle/deeper layers of the tissue in particular, appeared to have produced the protein and deposited it in the interterritorial matrix. The protein was neither seen in the superficial nor in the deepest regions of the articular cartilage. Based on its immunolocalization we have named this protein CILP (cartilage intermediate layer protein).     

Effects of parathyroid hormone (PTH) and PTH-related peptide on expressions of matrix metalloproteinase-2, -3, and -9 in growth plate chondrocyte cultures

The roles of PTH and PTH-related peptide (PTH-rp) in the expression of matrix metalloproteinases (MMPs) during endochondral bone formation were investigated, using various cartilages obtained from young rabbits and rabbit chondrocyte cultures. Immunohistochemical, immunoblotting, zymographical, and/or Northern blot analyses showed that MMP-2 and -9 levels were much higher in the growth plate than in permanent cartilage in vivo. In growth plate chondrocyte cultures, PTH, PTH-rp, and (Bu)2cAMP increased the amount of MMP-2 present in the culture medium, as revealed by zymograms and immunoblots, whereas the other tested growth factors or cytokines, including bone morphogenetic protein-2 and interleukin-1, did not increase the MMP-2 level. PTH also increased the MMP-2 messenger RNA level within 24 h. In addition, PTH increased MMP-3 and -9 levels in the growth plate chondrocyte cultures. However, in articular chondrocyte cultures, PTH had little effect on the levels of MMP-2, -3, and -9. In contrast to PTH, interleukin-1 induced MMP-3 and -9, but not MMP-2, in growth plate and articular chondrocytes. These findings suggest that in ossifying cartilage, PTH/PTH-rp plays a pivotal role in the induction of various MMPs, including MMP-2 (which is considered to be a constitutive enzyme), and that PTH/PTH-rp is involved in the control of cartilage-matrix degradation during endochondral bone formation.     

Effects of fibroblast growth factor-2 on longitudinal bone growth

In vivo, fibroblast growth factor-2 (FGF-2) inhibits longitudinal bone growth. Similarly, activating FGF receptor 3 mutations impair growth in achondroplasia and thanatophoric dysplasia. To investigate the underlying mechanisms, we chose a fetal rat metatarsal organ culture system that would maintain growth plate histological architecture. Addition of FGF-2 to the serum-free medium inhibited longitudinal growth. We next assessed each major component of longitudinal growth: proliferation, cellular hypertrophy, and cartilage matrix synthesis. Surprisingly, FGF-2 stimulated proliferation, as assessed by [3H]thymidine incorporation. However, autoradiographic studies demonstrated that this increased proliferation occurred only in the perichondrium, whereas decreased labeling was seen in the proliferative and epiphyseal chondrocytes. FGF-2 also caused a marked decrease in the number of hypertrophic chondrocytes. To assess cartilage matrix synthesis, we measured 35SO4 incorporation into newly synthesized glycosaminoglycans. Low concentrations (10 ng/ml) of FGF-2 stimulated cartilage matrix production, but high concentrations (1000 ng/ml) inhibited matrix production. We conclude that FGF-2 inhibits longitudinal bone growth by three mechanisms: decreased growth plate chondrocyte proliferation, decreased cellular hypertrophy, and, at high concentrations, decreased cartilage matrix production. These effects may explain the impaired growth seen in patients with achondroplasia and related skeletal dysplasias.     

IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses

IL-18, a cytokine originally identified as IFN-gamma-inducing factor, is a member of the IL-1 family of proteins. Because IL-1alpha and IL-1beta are important mediators in the pathogenesis of arthritis, the present study addresses the expression of IL-18 and its role in regulating in articular chondrocytes. IL-18 mRNA was induced by IL-1beta in chondrocytes. Chondrocytes produced the IL-18 precursor and in response to IL-1 stimulation secreted the mature form of IL-18. Studies on IL-18 effects on chondrocytes showed that it inhibits TGF-beta-induced proliferation and enhances nitric oxide production. IL-18 stimulated the expression of several genes in normal human articular chondrocytes including inducible nitric oxide synthase, inducible cyclooxygenase, IL-6, and stromelysin. Gene expression was associated with the synthesis of the corresponding proteins. Treatment of normal human articular cartilage with IL-18 increased the release of glycosaminoglycans. These finding identify IL-18 as a cytokine that regulates chondrocyte responses and contributes to cartilage degradation.     

Enhancement of SPARC (osteonectin) synthesis in arthritic cartilage. Increased levels in synovial fluids from patients with rheumatoid arthritis and regulation by growth factors and cytokines in chondrocyte cultures

OBJECTIVE: To investigate the roles of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) in arthritis, using cartilage and synovium specimens and synovial fluids (SF) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), and to examine the effects of cytokines, growth factors, and hormones on SPARC synthesis by chondrocytes in culture. METHODS: SPARC in cartilage and synovium was immunostained with monoclonal antibodies. SPARC synthesis by cultured chondrocytes was measured by Northern blot analysis, immunoblotting, and sandwich enzyme-linked immunosorbent assay. RESULTS: SPARC was identified in numerous chondrocytes in the superficial and middle zones and in regenerating chondrocytes of RA and OA joints, whereas such staining was absent in these zones of normal cartilage, except for weak signals from a few chondrocytes in the deep zone. In addition, SPARC synthesis was enhanced in synovial cells of RA and OA joints. The average SPARC level in SF was 10-fold higher in the RA than in the OA population. In rabbit articular chondrocyte cultures, administration of transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 increased SPARC levels at 24-48 hours, whereas interleukin-lbeta (IL-1 beta), IL-1 alpha, tumor necrosis factor alpha, lipopolysaccharide, phorbol myristate acetate, basic fibroblast growth factor, and dexamethasone decreased SPARC levels at 24-72 hours. TGF beta increased SPARC messenger RNA (mRNA) levels at 24 hours, whereas IL-1 beta caused a marked decrease in SPARC mRNA levels at 24 hours. Furthermore, IL-1 decreased the glycosylation of SPARC. CONCLUSION: These findings suggest that various growth factors and cytokines, including TGF beta 1 and IL-1 beta, regulate the production of SPARC by chondrocytes at pre- and posttranslational levels, and that SPARC synthesis is markedly enhanced in arthritic joints.