Oscillatory fluid flow regulates glycosaminoglycan production via an intracellular calcium pathway in meniscal cells.
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/microg cell protein) compared to the no-flow control (158 +/- 86 ng/microg). 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/microg). 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.
Department of Mechanical Engineering, Michigan Technological University, Houghton, Michigan 49931, USA.
Robert L
RL
Eifler
Eric R
ER
Blough
Jennifer M
JM
Dehlin
Tammy L
TL
Haut Donahue
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D040542:Mechanotransduction, Cellular / Q000187:drug effects / Q000502:physiology*; D008592:Menisci, Tibial / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D013314:Stress, Mechanical; D019284:Thapsigargin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D006025:Glycosaminoglycans; C013786:A73025; D019284:Thapsigargin; D002118:Calcium