Development of an organotypic microfluidic model to reproduce monocyte extravasation process in the osteoarthritic joint

Purpose: Osteoarthritis (OA) is a complex disease whereby synovitis can play a crucial role. One of the hallmarks of synovitis is the increased infiltration in synovium of monocytes/macrophages, which release pro-inflammatory mediators contributing to cartilage degradation. Hence, the recruitment of monocytes from the bloodstream and their extravasation into synovium represent important events in OA onset and progression. Here, we developed an organotypic 3D microfluidic model of the articular joint to investigate monocyte extravasation to the synovium, taking advantage of the ability of microfluidic systems to model dynamic biological processes in highly controlled microenvironments. Methods: A microfluidic chip mimicking the organization of the articular joint was designed in AutoCAD. The chip included cartilage and synovium compartments separated by a channel for synovial fluid. The synovium compartment integrated a microchannel for the generation of an endothelial monolayer to model the synovial postcapillary venule. Computational simulations (COMSOL Multiphysics) were carried out to determine shear stress distribution along the microchannel walls and the diffusion of chemokines from the synovial fluid to the synovium compartment. The microfluidic chip was fabricated via soft lithography techniques. Articular chondrocytes and synovial fibroblasts, obtained from biopsies of OA patients, were cultured in fibrin gel within cartilage and synovium compartments, respectively. Cell density and fibrin concentration were varied to select the best 3D culture conditions in terms of cell viability, morphology, and outgrowth by Live&Dead assay after 2 hours and 7 days. To resemble the endothelial wall of postcapillary venules, endothelial cells were seeded in the microchannel. The permeability of the endothelial monolayer was assessed by injecting 70 kDa FITC-dextran in the microchannel and analyzing its diffusion across the endothelial wall. Conversely, chemokine diffusion into the synovium compartment was evaluated by monitoring the diffusion of 10 kDa FITC-dextran injected in the synovial fluid compartment. Results: The computational simulations showed that the physiological shear stress necessary for the extravasation process (0.1 Pa) was achieved in the endothelialized microchannel using a flow rate lower than 60 μL/h. A cell concentration of 2.5 M/mL in 20 mg/mL fibrin gel was selected as the best 3D culture condition for articular chondrocytes and synovial fibroblasts. Indeed, higher cell densities caused relevant cell outgrowth from the gel and induced gel shrinkage. A fibrin concentration lower than 20 mg/mL caused cell outgrowth, while higher values resulted in inferior cell spreading. The endothelial cell concentration for the microchannel seeding was optimized at 10 M/mL. This condition permitted to obtain a confluent monolayer after 24 hours from injection. Lower and higher endothelial cell seeding densities determined respectively the non-formation of the monolayer and the formation of cell clusters blocking the microchannel. The integrity of the endothelial monolayer was demonstrated by the ability to hinder the diffusion of 70 kDa FITC-dextran across the endothelium, which was instead free to diffuse in constructs without endothelialization. We also verified that monolayer integrity was maintained under the flow rate selected from the computational analysis. Finally, experiments monitoring the diffusion of 10 kDa FITC-dextran in the synovium compartment yielded results coherent with the numerical model, showing that the endothelial monolayer determined chemokine accumulation in the synovium compartment. Conclusions: We designed and developed a new microfluidic model recapitulating the organization of the articular joint to investigate the biological mechanisms driving monocyte extravasation in the synovium. This platform will be used in the next future to test compounds targeting chemokine signaling axes responsible for monocyte recruitment.