Dynamic compression alters NFkappaB activation and IkappaB-alpha expression in IL-1beta-stimulated chondrocyte/agarose constructs.

OBJECTIVE AND DESIGN: Determine the effect of IL-1beta and dynamic compression on NFkappaB activation and IkappaB-alpha gene expression in chondrocyte/agarose constructs. METHODS: Constructs were cultured under free-swelling conditions or subjected to dynamic compression for up to 360 min with IL-1beta and/or PDTC (inhibits NFkappaB activation). Nuclear translocation of NFkappaB-p65 was analysed by immunofluoresence microscopy. Gene expression of IkappaB-alpha, iNOS, IL-1beta and IL-4 was assessed by real-time qPCR. RESULTS: Nuclear translocation of NFkappaB-p65 was concomitant with an increase in nuclear fluorescence intensity which reached maximum values at 60 min with IL-1beta (p < 0.001). Dynamic compression or PDTC reduced nuclear fluorescence and NFkappaB nuclear translocation in cytokine-treated constructs (p < 0.001 and p < 0.01 respectively). IL-1beta increased IkappaB-alpha expression (p < 0.001) at 60 min and either induced iNOS (p < 0.001) and IL-1beta (p < 0.01) or inhibited IL-4 (p < 0.05) expression at 360 min. These time-dependent events were partially reversed by dynamic compression or PDTC (p < 0.01) with IL-1beta. Co-stimulation by dynamic compression and PDTC favoured suppression (IkappaB-alpha, iNOS, IL-1beta) or induction (IL-4) of gene expression. CONCLUSIONS: NFkappaB is one of the key players in the mechanical and inflammatory pathways, and its inhibition by a biophysical/therapeutic approach could be a strategy for attenuating the catabolic response in osteoarthritis.

The effects of direct current stimulation on isolated chondrocytes seeded in 3D agarose constructs.

Endogenous electrical activity has been detected in articular cartilage. It has previously been suggested that the associated electrical currents and potentials are important to the mechanotransduction processes in cartilage. The present study investigates the effects of direct current on cell proliferation and matrix synthesis, using the well established 3D chondrocyte--agarose model system. Bovine chondrocytes isolated from metacarpalphalangeal joints were seeded in agarose constructs and exposed to a current density of 4 mA/cm2 for 6 h, a magnitude and period which was shown to maintain cell viability. The influence of the optimized electric stimulus was assessed by protein incorporation and mRNA measurements, using radiolabels and real-time QPCR, respectively. Results indicated no systematic influences of electrical current on protein synthesis, cell proliferation and mRNA expression levels. These data suggest that both the mode of stimulation and the model system are critical for the in vitro modulation of chondrocyte metabolism.

Dynamic compression influences interleukin-1beta-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression.

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.

Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs.

BACKGROUND: Nitric oxide and prostaglandin E2 (PGE2play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1beta and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway. METHODS: Chondrocyte/agarose constructs were cultured under free-swelling conditions with or without IL-1beta and/or SB203580 (inhibitor of p38 MAPK) for up to 48 hours. Using a fully characterized bioreactor system, constructs were subjected to dynamic compression for 6, 12 and 48 hours under similar treatments. The activation or inhibition of p38 MAPK by IL-1beta and/or SB203580 was analyzed by western blotting. iNOS, COX-2, aggrecan and collagen type II signals were assessed utilizing real-time quantitative PCR coupled with molecular beacons. Release of nitrite and PGE2 was quantified using biochemical assays. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test were used to examine data. RESULTS: IL-1beta activated the phosphorylation of p38 MAPK and this effect was abolished by SB203580. IL-1beta induced a transient increase in iNOS expression and stimulated the production of nitrite release. Stimulation by either dynamic compression or SB203580 in isolation reduced the IL-1beta induced iNOS expression and nitrite production. However, co-stimulation with both dynamic compression and SB203580 inhibited the expression levels of iNOS and production of nitrite induced by the cytokine. IL-1beta induced a transient increase in COX-2 expression and stimulated the cumulative production of PGE2 release. These effects were inhibited by dynamic compression or SB203580. Co-stimulation with both dynamic compression and SB203580 restored cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response with the cytokine and/or SB203580. CONCLUSION: These data suggest that dynamic compression directly influences the expression levels of iNOS and COX-2. These molecules are current targets for pharmacological intervention, raising the possibility for integrated pharmacological and biophysical therapies for the treatment of cartilage joint disorders.