| Summary: | Osmotic loading of articular cartilage has been used to study cell-tissue interactions and mechanisms in chondrocyte volume regulation <em>in situ</em>. Since cell volume changes are likely to affect cell’s mechanotransduction, it is important to understand how environmental factors, such as composition of the immersion medium and temperature affect cell volume changes <em>in situ</em> in osmotically challenged articular cartilage. In this study, chondrocytes were imaged <em>in situ</em> with a confocal laser scanning microscope (CLSM) through cartilage surface before and 3 min and 120 min after a hypo-osmotic challenge. Samples were measured either in phosphate buffered saline (PBS, without glucose and Ca<sup>2+</sup>) or in Dulbecco’s modified Eagle’s medium (DMEM, with glucose and Ca<sup>2+</sup>), and at 21 °C or at 37 °C. In all groups, cell volumes increased shortly after the hypotonic challenge and then recovered back to the original volumes. At both observation time points, cell volume changes as a result of the osmotic challenge were similar in PBS and DMEM in both temperatures. Our results indicate that the initial chondrocyte swelling and volume recovery as a result of the hypo-osmotic challenge of cartilage are not dependent on commonly used immersion media or temperature.
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