Response of sheep chondrocytes to changes in substrate stiffness from 2 to 20 Pa: effect of cell passaging.

AIM

The influence of culture substrate stiffness (in the kPa range) on chondrocyte behavior has been described. Here we describe the response to variations in substrate stiffness in a soft range (2-20 Pa), as it may play a role in understanding cartilage physiopathology.

METHODS

We developed a system for cell culture in substrates with different elastic moduli using collagen hydrogels and evaluated chondrocytes after 2, 4, and 7 days in monolayer and three-dimensional (3D) cultures. Experiments were performed in normoxia and hypoxia in order to describe the effect of a low oxygen environment on chondrocytes. Finally, we also evaluated if dedifferentiated cells preserve the capacity for mechanosensing.

RESULTS

Chondrocytes showed less proliferating activity when cultured in monolayer in the more compliant substrates. Expression of the cartilage markers Aggrecan (Acan), type II collagen (Col2a1), and Sox9 was upregulated in the less stiff gels (both in monolayer and in 3D culture). Stiffer gels induced an organization of the actin cytoskeleton that correlated with the loss of a chondrocyte phenotype. When cells were cultured in hypoxia, we observed changes in the cellular response that were mediated by HIF-1α. Results in 3D hypoxia cultures were opposite to those found in normoxia, but remained unchanged in monolayer hypoxic experiments. Similar results were found for dedifferentiated cells.

CONCLUSIONS

Chondrocytes respond differently according to the stiffness of the substrate. This response depends greatly on the oxygen environment and on whether the chondrocyte is embedded or grown onto the hydrogel, since mechanosensing capacity was not lost with cell expansion.