Effects of growth factors and interleukin-1 alpha on proteoglycan and type II collagen turnover in bovine nasal and articular chondrocyte pellet cultures

Xu, Chi

doi:10.1210/en.137.8.3557

Cited by 33 publications

(22 citation statements)

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“…A simple method for maintaining the cartilage phenotype in chondrocytes in culture is the use of pellet cultures, in which freshly isolated cells are centrifuged and maintained in polypropylene centrifuge tubes where they form a continuous matrix (Xu et al, 1996). In this way, chondrocytes maintain a high density and do not adhere to the untreated plastic, thereby retaining their three-dimensional (3D) conformation.…”

Section: Introductionmentioning

confidence: 99%

Role of pericellular matrix in development of a mechanically functional neocartilage

Graff¹,

Kelley²,

Lee³

2003

Biotech & Bioengineering

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The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.

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Section: Introductionmentioning

confidence: 99%

Role of pericellular matrix in development of a mechanically functional neocartilage

Graff¹,

Kelley²,

Lee³

2003

Biotech & Bioengineering

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“…An increase in the rate of proteoglycan degradation and decreased proteoglycan synthesis are among its most prominent catabolic actions in diseased articular cartilage (22)(23)(24). IL-1 can also induce cartilage nonresponsiveness to IGF-I through a mechanism that remains unknown (22,25).…”

mentioning

confidence: 99%

Regulation of Insulin-Like Growth Factor-Binding Protein-5 by Insulin-Like Growth Factor I and Interleukin-1 in Ovine Articular Chondrocytes

Sunic

1998

Endocrinology

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Insulin-like growth factors (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes' responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1-3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR 3 IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22-and 16-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1-3)IGF-I or LR 3 IGF-I. Basic fibroblast growth factor, transforming growth factor-␤, and plateletderived growth factor had no effect on OAC IGFBPs. However, IL-1␣ increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1␣, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1␣ resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two factors. Des(1-3)IGF-I and LR 3 IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1␣ reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1␣ were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1␣ synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1␣ in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides. (Endocrinology 139: 2356(Endocrinology 139: -2362(Endocrinology 139: , 1998

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“…12 Pellet-cultured chondrocytes form cartilage-specific ECM by expression of proteoglycans and collagen type II. 12,13 This 3-D ECM reflects the specific cellular environment of the chondrocytes, allowing functional interactions between the cells and their ECM. For co-cultivation, 2 SV40T antigen-transformed human SF cell lines are available: 1) The hepatic sinusoidal endothelium (HSE) cell line was derived from an RA patient and shows an aggressive invasive behavior in vitro, whereas 2) the K4IM cell line was established from a healthy donor and represents the noninvasive phenotype.…”

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confidence: 99%

Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

et al. 2007

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The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBi™-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time. (Journal of Biomolecular Screening 2007:956-965)

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Effects of growth factors and interleukin-1 alpha on proteoglycan and type II collagen turnover in bovine nasal and articular chondrocyte pellet cultures

Cited by 33 publications

References 0 publications

Role of pericellular matrix in development of a mechanically functional neocartilage

Role of pericellular matrix in development of a mechanically functional neocartilage

Regulation of Insulin-Like Growth Factor-Binding Protein-5 by Insulin-Like Growth Factor I and Interleukin-1 in Ovine Articular Chondrocytes

Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

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