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

Introduction

Mechanical stimulation of chondrocytes plays essential roles in the homeostasis of cartilages and arthritic disease. Within the knee cartilage, mechanical stimulation is the main mediator of intercellular communication as it is devoid of any blood vessels or nerves. Diffusion of signaling molecules and nutrients thus occurs through load-induced extracellular fluid flow. The bulk of the cartilage is made of interstitial matrix which absorbs most of the compressive and sliding forces exerted during movement. A softer layer, the pericellular matrix then surrounds the chondrocytes and protects them from forces exerted onto the interstitial layer. During movement, chondrocytes sense extracellular matrix deformation through these two successive layers. To explore the impact of mechanical stimulation, cartilage-on-chip models have been developed incorporating a mechanical actuation unit.

Here, Paggi et al. have implemented a cartilage-on-chip platform which can generate both compressive and multi-directional mechanical stimulations on chondrocyte-laden hydrogels. Hence, chondrocytes can experience healthy and/or hyper-physiological loading as the joint would do during movement depending on the amount of applied load. Collectively, their results highlight the importance of applying proper mechanical stimulation for proper regulation of chondrocytes and reproducing their microenvironment through the production of native pericellular matrix.

Experimental procedure

The cartilage-on-chip platform is composed of a central cell culture chamber caught in between a perfusion channel and a mechanical actuation unit. The actuation unit is made of a 50µm thick PDMS membrane actuated by three individual chambers connected to each other. Cells are encapsulated within an agarose hydrogel in the central chamber and can be mechanically stimulated by the deflection of the PDMS membrane. Pressure is generated using Fluigent positive pressure controller MFCS-EZ. When the same positive pressure is applied in the three actuation chambers, the compression is homogeneous. Different deformations patterns can be obtained using a dedicated sequence of positive and negative pressures in the different actuation chambers, giving rise to a multi-directional mechanical stimulation (mdms). This mdms pattern was shown to consist of a combination of compressive and shear forces, and is reminiscent of a waveform of mechanical stimulation, mimicking the rolling motion of two cartilage surfaces.

Cartilage-on-chip design (from Paggi et al. 2022)

a) Schematic representation of the knee joint. b) Design of the cartilage-on-chip device, highlighting its main features. c) Top view of the cartilage-on-chip device filled in with food dyes for visualization purposes – actuation unit in blue; cell-hydrogel chamber in red and perfusion channel in yellow. d) Side-view schematic of the joint during motion, depicting the generated multi-directional mechanical stimulation (shear strain – green arrow & compression – blue arrow). e i–iii) Top view of the cartilage-on-chip device filled in with human chondrocytes in agarose exposed to a sequence generating multi-directional mechanical stimulation (blue arrows depict compression and green arrows shear strain).

Results

Chondrocytes embedded in agarose hydrogel within the cartilage-on-chip device displayed a pro-inflammatory response (cytokine release) which was further amplified by mechanical actuation. Exposure to mechanical stimulation and especially to an mdms pattern was found to have an impact on gene expression of chondrocytes markers. 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. 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 mdms stimulation. Finally, mechanical cues enhanced the production of essential cartilage ECM markers, such as aggrecan, collagen II and collagen VI, a marker for the pericellular matrix around the chondrocytes.

Effect of various mechanical stimulation patterns (compressive forces only or multi-directional mechanical stimulation) on chondrocyte functions within the cartilage-on-chip platform

Both mechanical stimulation options promote the production of extracellular matrix by the chondrocytes. Pericellular matrix production around every single cell was similar for both mechanical stimulation strategies (blue ring shown on histology sections), while mdms was found to clearly promote the production and distribution of the interstitial matrix across the entire tissue, as indicated by the blue homogeneous color. Mechanical stimulation of the chondrocytes thereby allows emulating the native articular cartilage microenvironment, with a clear advantage of mdms stimuli.