Matrix Viscoelasticity Regulates Dermal Fibroblast Activation in a Three-Dimensional Fibrillar Microenvironment

Abstract Purpose Fibrosis is the pathological remodeling of the extracellular matrix (ECM) that is largely orchestrated by activated fibroblasts. The mechanical properties of the ECM change drastically during fibrosis, and fibroblasts become increasingly activated by mechanical environments that mimic the properties of fibrotic tissues. While the effects of increased elastic modulus (stiffness) on fibroblast activation have been well-studied, the impact of changes in viscoelasticity is less clear. Here, we sought to determine how fibroblast activation is altered by changes in viscoelasticity in a three-dimensional, fibrillar microenvironment. Methods We employed 3D alginate collagen I hydrogels with independently tunable stiffness and stress relaxation rates. Dermal fibroblasts were encapsulated in hydrogels with four distinct mechanical profiles (soft: 3 kPa or stiff: 10 kPa, fast stress relaxing: τ 1/2 ≈ 160 s or slow stress relaxing: τ 1/2 ≈ 1600 s). We assessed fibroblast activation by changes in cell morphology, expression of key activation markers, and evidence of ECM remodeling. Results Fibrillar alginate collagen networks enhanced fibroblast spreading, α-smooth muscle actin stress fiber formation, and fibroblast activation protein-α expression in matrices that were slow relaxing or stiff. The presence of the fibrillar network further enhanced fibroblast activation, independent of the changes driven by matrix viscoelasticity. ECM remodeling was also promoted by slow relaxing matrices, with increased fibronectin deposition and more remodeling of the local collagen fiber network. Conclusions Our results demonstrate that fibroblast activation is highly responsive to matrix stress relaxation rate, and that models incorporating fibrillar, viscoelastic networks can provide new insights into the role of ECM mechanics driving fibroblast activation.