Abstract
Chondrocyte sensitivity to strain depends on signal transduction pathways which include integrin-dependent increases in intracellular calcium. Human articular chondrocytes were cultured as monolayers in silicone dishes. After loading the cells with the calcium-fluorescent dye Fluo-3/AM the dishes were mounted in a 4-point bending apparatus and then fixed to a laser scanning confocal microscope. Biaxial substrate strain (15 000e) was applied to the silicone dish via a hand operated cam rotated at ~60 RPM (1 Hz) for 10 or for 50 cycles. Changes in intracellular calcium in single cells were determined by measuring the mean pixel values in the basal and stimulated images taken at different time points. The data reported for 50 cycle treatments represent 49 single cells of six independent cell isolations. The data for 10 cycle strain treatment are from a single experimental setup.
Increases in intracellular calcium were consistently observed in chondrocytes exposed to 15 000me for 50 cycles in a range from 1.3- to 4.0-fold with an average of 2.3-fold (SD=0.79). Few cells responded before 30 minutes but most of the responses occurred 30–60 minutes after strain. Consistent intracellular Ca++-increases were also seen after 10 strain cycles, however responses were detected within 5 minutes post-strain. The relative increase (2.7-fold ± 1.7) was similar in magnitude to 50 cycle responses.
Intracellular Ca++-fluxes in chondrocytes and other cells occur by at least two different mechanisms: through stretch-activated channels in the plasma membrane permit immediate Ca++-influx during strain application or by Ca++-efflux from intracellular compartments stimulated by slower acting second messengers. Our results suggest that the early response to 10 strain cycles is due to Ca++-influx via membrane channels while the later response to 50 cycles is due to Ca++-efflux from intracellular compartments, probably mediated by cytokines released in response to an initial Ca++-influx from the medium.
