Matrix Adhesiveness Regulates Myofibroblast Differentiation from Vocal Fold Fibroblasts in a Bio-orthogonally Cross-linked Hydrogel

Repeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant in vitro model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (trans-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz-Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz-TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-β1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-β1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.