Abstract Twenty percent of all primary care consults are related to musculoskeletal diseases; 30% of these are associated with tendinopathies. Pathogenesis of tendinopathy includes increased inflammatory signaling and extracellular matrix (ECM) remodeling. This remodeling leads to softer tendinopathic tendons, increasing the risk of tearing. Yet the relative roles of chronic inflammation and ECM stiffness in the initiation and progression of tendon disease remain controversial and are difficult to decouple in patient populations. We and others have shown that, in 2D cell culture using interleukin-1β (IL-1β) as a stimulant, patient-derived tendinopathic fibroblasts exhibit a stronger inflammatory response that is further enhanced on soft substrates. This inflammatory response is dependent on NF-κB signaling, which we have previously established as a critical regulator of tendon disease and healing. Yet these studies are limited by the use of classical 2D culture approaches and fail to recapitulate in vivo cell behavior or provide insight into ECM remodeling. The ability to visualize cytokine receptor clustering in 3D environments has further demonstrated that cellular sensitivity to cytokines is based on the properties of the ECM. Although these studies suggest physicochemical coupling between ECM stiffness (physical) and inflammatory signaling (chemical) that sustains chronic loss of tendon mechanical function, the mechanisms of how ECM drives cell behavior in 3D tissues like tendon remain unknown. Therefore, there remains a critical need to define the physicochemical cell-ECM interactions that regulate tendon function to discover the mechanisms underlying tendinopathies and treatments. Our long-term goal is to develop therapeutic strategies for the clinical treatment of tendinopathy by identifying key cell-ECM mechanisms driving chronic inflammatory tendon disease. Our overall objective in this application is to develop a novel approach to studying the physicochem