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

Abstract

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 celladhesive 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.

Modeling the Maturation of the Vocal Fold Lamina Propria Using a Bioorthogonally Tunable Hydrogel Platform

“…With an overarching goal of modeling vocal fold maturation, we exploited the inverse electron demand Diels-Alder reaction between s-tetrazine and strained alkenes to develop a bioorthogonal hydrogel platform that is cell adhesive, enzymatically degradable, and dynamically tunable, allowing user-directed matrix stiffening in a spatiotemporal manner in the presence of MSCs. Instead of using two different types of crosslinking reactions, [16,28] we utilized slow (Nb) and fast (Tz) dienophiles to facilitate 3D cell encapsulation, ensure close to 100% conjugation of RGD to the network [14] and enable matrix stiffening in a diffusioncontrolled manner without the need for additional reagents, catalysts, or environmental changes (pH or temperature) that are potentially detrimental to cells or interfere with normal cell functions. [13,[29][30][31][32][33] Because diffusion of HA-TCO through the crosslinked network is relatively slow, our platform closely mimics the gradual changes in matrix stiffness during tissue maturation.…”

“…Based on our previous work, RGD conjugation in the primary network is close to 100%. [14] The mechanical properties of various HA gels were analyzed macroscopically using a DMA and microscopically using a custom micromechanical tester. Under unconfined compression, nLP, mLP, and sLP gels exhibited an average compression modulus of 4.9 ± 0.6 kPa, 6.1 ±1.1 kPa, and 10.3 ± 3.0 kPa, respectively.…”

“…The relatively slow Tz/Nb ligation allowed the establishment of the initial covalent network, while the rapid and efficient Tz/TCO reaction enabled modification of the cell‐laden construct (specifically matrix adhesiveness) in a time‐delayed manner. [ 14,15 ] Herein, we further exploit the utility of this platform in modeling the vocal fold development. A homogeneously soft matrix was established via Tz/Nb reaction with a 5/2 stoichiometry.…”

A Promotion Role of MIR31 in the Process of Vocal Fold Wound Healing

“…Our results suggested that MIR31 may co-function with the 17 collagen production regulators to play a role in the VFWH process. In addition, our study also showed that MIR31 was related to multiple biological processes that were linked to wound healing, including fibrosis and keloid [ 31 , 32 ].…”

Hydrogels to Recapture Extracellular Matrix Cues That Regulate Vascularization

“…To overcome the limitations of traditional hydrogels, researchers have explored complementary approaches that confer defined cell-guidance cues and spatiotemporal dynamic modulation. [30][31][32] Using modular and orthogonal design strategies, 33,34 natural polysaccharides and proteins found in ECM can be modified to generate hydrogel with defined chemical and mechanical environments. These engineered hydrogel platforms have been shown to allow for temporal monitoring of sequential cellular events during vascular morphogenesis and vascularization, establishing engineered microvasculature and perfusable capillary networks in vitro and in vivo.…”