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

Toward the goal of establishing an engineered model of the vocal fold lamina propria (LP), mesenchymal stem cells (MSCs) were encapsulated in hyaluronic acid (HA)-based hydrogels employing tetrazine ligation with strained alkenes. To mimic matrix stiffening during LP maturation, diffusion-controlled interfacial bioorthogonal crosslinking was carried out on the soft cellular construct using HA modified with a ferocious dienophile, trans-cyclooctene (TCO). Cultures were maintained in MSC growth media for 14 days to afford a model of a newborn LP that is homogeneously soft (nLP), a homogeneously stiffened construct zero (sLP0) or 7 days (sLP7) post cell encapsulation, and a mature LP model (mLP) with a stiff top layer and a soft bottom layer. Installation of additional HA crosslinks restricted cell spreading. Compared to the nLP controls, sLP7 conditions upregulated the expression of fibrous matrix proteins (Col I, DCN, and FN EDA), classic fibroblastic markers (TNC, FAP, and FSP1), and matrix remodeling enzymes (MMP2, TIMP1, and HAS3). Day 7 stiffening also upregulated the catabolic activities, enhanced ECM turnover, and promoted YAP expression. Overall, in situ delayed matrix stiffening promoted a fibroblast transition from MSCs and enhanced YAP-regulated mechanosensing. This article is protected by copyright. All rights reserved.