Ion-channel Regulation of Chondrocyte Matrix Synthesis in 3D Culture Under Static and Dynamic Compression
Inhibition of various ion channels alters chondrocyte mechanotransduction in monolayer, but the mechanisms involved in chondrocyte mechanotransduction in three- dimensional culture remain unclear. The objective of this study was to investigate the effects of inhibiting putative ion-channel influenced mechanotransduction mechanisms on the chondrocyte responses to static and dynamic compression in three-dimensional culture. Bovine articular cartilage explants were used to investigate the dose-dependent inhibition and recovery of protein and sulfated glycosaminoglycan (sGAG) syntheses by four ion-channel inhibitors: 4-Aminopyridine (4AP), a K+ channel blocker; Nifedipine (Nf), a Ca2+ channel blocker; Gadolinium (Gd), a stretch-activated channel blocker; and Thapsigargin (Tg), which releases intracellular Ca2+ stores by inhibiting ATP-dependent Ca2+ pumps. Chondrocyte-seeded agarose gels were used to examine the influence of 20 h of static and dynamic loading in the presence of each of the inhibitors. Overall, treatment with the ion-channel inhibitors had a greater effect on sGAG synthesis, with the exception of Nf, which more substantially affected protein synthesis. Treatment with Tg significantly impaired both overall protein and sGAG synthesis, with a drastic reduction in sGAG synthesis. The inhibitors differentially influenced the responses to mechanical stimuli. Dynamic compression significantly upregulated protein synthesis but did not significantly affect sGAG synthesis with Nf or Tg treatment. Dynamic compression significantly upregulated both protein and sGAG synthesis rates with Gd treatment. There was no significant stimulation of either protein or sGAG synthesis by dynamic compression with 4AP treatment. Interruption of many ion-channel signaling mechanisms affected sGAG synthesis, suggesting a complicated, multi-pathway signaling process. Also, Ca2+ signaling may be critical for the transduction of mechanical stimulus in regulating sGAG synthesis. This modulation potentially occurs through direct interactions with the extracellular matrix.