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Abstract

Small proteoglycans (PGs) are supposed to play great roles in the assembly of cartilage matrix but the influence of cytokines and growth factors on their synthesis by articular chondrocytes is largely unknown. We investigated whether IL-1 and TGFbeta1 influence the production of small leucine-rich proteoglycans by chondrocytes cultured in a three-dimensional gel, as compared to the common monolayer system. Rabbit articular chondrocytes were cultured in alginate beads for 14 days or as monolayers for 7 days. The effect of 2 ng/ml IL-1beta or TGFbeta1 during the last two days in culture was determined, after [35S]methionine labeling over the last 24 h. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment of alginate beads whereas medium and cell-layer fractions were isolated from monolayer cultures. Total newly synthesized PGs were first isolated by anion-exchange chromatography and the small PGs were further separated from aggrecans by gel-filtration (Sepharose CL-4B) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Addition of TGFbeta1 resulted in an overall rise in neosynthesized small PG content in both culture systems. However, TGFbeta1 significantly increased to the same extent the percentage of small PGs laid down in the cell-associated and the further-removed matrix compartments of the beads culture (+60%) whereas it augmented the content of small PGs in the medium (+40%) and reduced that of the cell fraction (-35%) in the monolayer culture. By adding IL-1, the amount of total newly synthesized small PGs was decreased in monolayers while it increased in alginate beads. IL-1 was also shown to change the relative distribution of these molecules in the monolayer system in contrast to the alginate beads culture where the proportions were not significantly altered. Electrophoretic analysis of the 35S-labeled small PGs-containing fractions confirmed these effects at the level of the 45-50 kDa-related core proteins. This study demonstrates that TGFbeta and IL-1 differently influence small PG synthesis of rabbit articular chondrocytes depending on whether they are cultured in alginate beads or in monolayers. Moreover, the regulation of small PG expression appears to be different from that of high-molecular weight aggrecans. As these small molecules are playing major roles in matrix assembly and growth factor regulation, the data may have great relevance to the pathogenesis of osteoarthritis and repair of articular cartilage lesions.

Inflammation Induces Irreversible Biophysical Changes in Isolated Nucleus Pulposus Cells

“…A potential limitation of the current study is that the use of 2D culture may alter the NP cell phenotype [44], and may differentially regulate the inflammatory response [45]. However, NPs are known to respond to cytokines both in vivo and in vitro [20], and our findings on biophysical responses are assessed on cells in 3D-like rounded morphology.…”

Poster 245: In Vitro Model of Cartilage Degradation With Chondrocytes in a Three-Dimensional Culture System–Time-Dependent Involvement of MMP-3 and TIMP-1, -2 and -3

“…These observations have led to evaluations of cartilage destruction using IL-1β or TGF-β. Concerning culturing conditions, Demoor-Fossard et al (30) treated chondrocytes in culture with these cytokines, noted differences in ECM formation between monolayer and three-dimensional cultures, and reported that responses to biological stimuli such as cytokines similar to those of cartilage tissue were observed in three-dimensional cultures. This suggests that the responses observed in the ECM in monolayer cultures may not necessarily reflect physiologic responses.…”

Inhibition of toll-like receptor 4 protects against inflammation-induced mechanobiological alterations to intervertebral disc cells

“…Potential limitations of the study include the use of cell plating for expansion, which may alter NP cell phenotype ( Horner et al , 2002 ) as well as inflammatory response ( Demoor-Fossard et al , 1999 ). However, for osmotic and cytoskeletal analysis, NP cells were observed in a rounded morphology that more closely mimicked the 3D morphology of NP cells in situ .…”