Mechanical stimulation is a key component in the development of pericellular matrix around human chondrocytes

Abstract

Purpose: Articular cartilage within the joint experience multi-directional mechanical stimulation. These stimuli are transferred to the chondrocytes through the pericellular matrix, a small shell surrounding the cells. The signal is then translated by the cell, which subsequently responds by releasing catabolic or anabolic markers. Our aim was to be able to determine if, depending on the mechanical stimulus applied onto the chondrocytes, there would be a variation in extracellular matrix production. To address this, we used our cartilage-on-chip and studied the effects of mechanical stimulation on human articular chondrocytes. The cartilage-on-chip system includes a 3D cell-hydrogel compartment, a perfusion channel to provide nutrients and a mechanical actuation unit. The mechanical actuation unit, which is separated from the rest of the system by a thin elastic polydimethylsiloxane membrane, comprises 3 individually addressable chambers were positive or negative pressure can be applied (Fig. 1). We report here how different mechanical stimuli: 1) affect IL-6 cytokine production; and 2) the chondrocyte’s phenotype compared to static conditions; 3) influence the directionality in extracellular matrix deposition; 4) stimulate pericellular matrix formation, and; 5) differentially affect overall protein expression. Methods: The cartilage-on-chip was fabricated using soft-lithography with polydimethylsiloxane. Application of positive and/or negative pressure resulted in different sets of stimulations onto the 3D hydrogel (compression or compression & shear strain). Low melting temperature agarose (2% w/v) was used to embed human primary chondrocytes (hCHs), which were cultured in the platform for one day with proliferation medium followed by chondrocyte differentiation medium. First, the cell-laden constructs were stimulated for 7 days (1 h a day at 1 Hz) with either compression, or compression and shear strain (wave form) or without mechanical stimuli. Medium was collected from the device at day 1, 2, 3, 5 and 7 and IL-6 concentration quantified using enhanced SPRi. qPCR was performed at day 7 for Collagen type I and type II. Next, cell-laden hydrogels were cultured for 7 days in static condition and subsequently stimulated for 7 days with the two aforementioned mechanical actuation patterns, or in static conditions for a total of 14 days. Samples were fixed and stained for aggrecan, collagen type II and collagen type VI. Results: The expression of COL2A1 mRNA was higher in the mechanically stimulated samples in particular when using a combination of compression and shear strain. Interestingly, COL1A1 mRNA expression was inversely correlated with the COL2A1 mRNA with highest expression in the static conditions. Consequently, these results suggest the involvement of mechanical stimulation on the transition of human chondrocytes towards a more hyaline like cartilage phenotype. IL-6 secretion decreased over time with no detectable signal at day 7 for the static conditions indicating chondrocyte adaptation to the 3D environment. Moreover, IL-6 was higher in the constructs exposed to mechanical stimuli, indicating a role of mechanical stimulation in IL-6 production. Aggrecan, collagen type II and collagen type VI immunostaining revealed higher pericellular matrix production in dynamic conditions. The pericellular matrix formed was mainly present in the direction of the mechanical stimulation suggesting cell adaptation to the applied stimuli (Fig. 3). Furthermore, the combination of compression and shear strain additionally enhanced both aggrecan and collagen production. Conclusions: Our cartilage-on-a-chip platform allows exposure of hydrogel embedded chondrocytes to multi-modal mechanical stimuli. Mechanical actuation supported the creation of a pericellular matrix surrounding the cells. Here, chondrocytes responded differently both at the gene and protein levels depending on the stimuli applied. Interestingly, chondrocytes produced their matrix in the direction of the stimuli suggesting a cell adaptation or “self-protection” mechanism. We are currently investigating if, by applying different biochemical stimuli, it is possible to influence the composition of the pericellular matrix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)