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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. [ABSTRACT FROM AUTHOR]

• Monolithic device with a vertical membrane for mechanical stimulation of tissues. • Actuation unit with 3 individual chambers to apply negative and/or positive pressure. • Real-time tissue imaging at the single cell level: monitoring cell deformation. • Application of healthy and hyper-physiological compressions to a cartilage model. • Possible generation of combinations of compression and bulk shear. Movement is essential to our quality of life, and regulates cell behavior via mechanical stimulation. Here, we report a monolithic microfluidic platform, in which engineered tissues composed of cells in a hydrogel are exposed to gradients of mechanical compression. Mechanical stimulation is applied through the deflection of a thin polydimethylsiloxane (PDMS) vertical membrane. The device design and all actuation parameters were optimized in this work to produce physiologically relevant compression on a cartilage model (strain of 5–12 %), as well as gradients of compression ranging from healthy to hyper-physiological conditions in the same device, as evidenced by the measured gradients in cell deformation. While this work focuses on mechanical compression of engineered tissues, we also demonstrated that our platform allowed creating more sophisticated multi-modal stimulation patterns. As the membrane is actuated by three independently addressed yet connected pressurized chambers, a variety of programmable deflection patterns and various cell stimulation modalities can easily be created by tuning the pressure applied in the different chambers (positive vs. negative, and amplitude). Advantageously, the fabrication of this monolithic platform is straightforward, with a single-step process. Moreover, the vertical membrane configuration allows for real-time imaging of cells encapsulated in the hydrogel matrix. The herein reported platform is highly versatile and of great interest to model other types of tissues, which also experience complex mechanical actuation patterns in vivo. [ABSTRACT FROM AUTHOR]