Emulating the chondrocyte microenvironment using multi-directional mechanical stimulation in a cartilage-on-chip.

The multi-directional mechanical stimulation experienced by articular cartilage during motion is transferred to the chondrocytes through a thin layer of pericellular matrix around each cell; chondrocytes in turn respond by releasing matrix proteins and/or matrix-degrading enzymes. In the present study we investigated how different types of mechanical stimulation can affect a chondrocyte's phenotype and extracellular matrix (ECM) production. To this end, we employed a cartilage-on-chip system which allows exerting well-defined compressive and multi-directional mechanical stimulation on a 3D chondrocyte-laden agarose hydrogel using a thin deformable membrane and three individually addressed actuation chambers. First, the 3D chondrocyte culture in agarose responded to exposure to mechanical stimulation by an initial increase in IL-6 production and little-to-no change in IL-1β and TNF-α secretion after one day of on-chip culture. Exposure to mechanical stimulation enhanced COL2A1 (hyaline cartilage marker) and decreased COL1A1 (fibrotic cartilage) expression, this being more marked for the multi-directional stimulation. Remarkably, the production of glycosaminoglycans (GAGs), one of the main components of native cartilage ECM, was significantly increased after 15 days of on-chip culture and 14 days of mechanical stimulation. Specifically, a thin pericellular matrix shell (1-5 μm) surrounding the chondrocytes as well as an interstitial matrix, both reminiscent of the in vivo situation, were deposited. Matrix deposition was highest in chips exposed to multi-directional mechanical stimulation. Finally, exposure to mechanical cues enhanced the production of essential cartilage ECM markers, such as aggrecan, collagen II and collagen VI, a marker for the pericellular matrix. Altogether our results highlight the importance of mechanical cues, and using the right type of stimulation, to emulate in vitro , the chondrocyte microenvironment.