Oscillatory fluid flow regulates glycosaminoglycan production via an intracellular calcium pathway in meniscal cells

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Mechanical loading in the form of oscillatory fluid flow-induced shear stress was applied to meniscal cells while the biochemical response [intracellular calcium mobilization and sulfated glycosaminoglycan (GAG) production] was studied. Isolated rabbit meniscal cells were cultured onto microscope slides and placed in a parallel plate flow chamber. Cells were exposed to oscillating fluid flow-induced shear stresses of 4 Pa for sulfated GAG studies and 0-6.5 Pa for calcium studies. The calcium response was monitored using a fluorescent probe and imaging techniques, sulfated GAG production was measured using the modified 1,9-dimethylmethylene blue method, and thapsigargin was used to block intracellular calcium ([Ca2+]i) mobilization. A significant dose-dependent relationship was found for the percentage of cells responding to oscillating fluid flow with an increase in [Ca2+]i versus shear stress level. The percentage of cells responding decreased linearly from 72% ± 17% at 6.5 Pa to 28% ± 7% at 2.0 Pa to 2% ± 1% for baseline no-flow (0 Pa). No differences were found in the amplitude of the calcium response of responding cells for any shear stress level. Oscillating fluid flow-induced shear stresses of 4 Pa produced a significantly greater amount of sulfated GAGs (253 ± 95 ng GAG/μg cell protein) compared to the no-flow control (158 ± 86 ng/μg). The addition of thapsigargin to the media inhibited both the intracellular calcium response to oscillating fluid flow (less than 1.5% of the cells responded) and the increase in GAG production following oscillating fluid flow, which was returned to control levels (170 ± 72 ng/ μg). These findings suggest that oscillatory fluid flow-induced shear stress increases intracellular calcium levels and sulfated GAG production. Furthermore, they suggests that calcium may modulate the biochemical pathway that leads to sulfated GAG production. © 2005 Orthopaedic Research Society.

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Journal of Orthopaedic Research