A bioorthogonal hydrogel platform for the development of an engineered vocal fold tissue model

Journal Title
Journal ISSN
Volume Title
University of Delaware
Vocal fold lamina propria, with a complex multilayered structure, exhibits optimal biomechanical properties for voice production. Vocal fold scarring disrupts the laminated structure, alters the tissue viscoelasticity, and severely compromises the phonatory function. Therapeutic interventions are limited because of the lack of understanding of molecular mechanisms contributing to vocal fold scarring. The situation is exacerbated due to the scarcity of healthy human tissues, inability of animal models to recapitulate the human physiology, and unavailability of in vitro physiologically relevant tissue models. ☐ The goal of this dissertation is to develop a bioorthogonal hydrogel platform for the engineering of a human vocal fold tissue model. To this end, hydrogel precursors tagged with bioorthogonal tetrazine (Tz), trans-cyclooctene (TCO), norbornene (Nb), thiol (SH) or acrylate (AC) groups were synthesized using hyaluronic acid, synthetic polymers, and bioactive peptides. Various dienophiles with different reactivities toward tetrazine were conjugate to matrix metalloprotease (MMP)-degradable peptides, integrin-binding RGD peptide, and basic fibroblast growth factor-mimetic peptide. The composition and purity of these conjugates were characterized by proton nuclear magnetic resonance (1HNMR), high performance liquid chromatography (HPLC), mass spectrometry (MS), and ultraviolet-visible spectroscopy (UV-vis). ☐ Using the modular building blocks, a lamina propria-mimetic construct with a layered structure was developed. Taking advantage of the exceptional rate of the Tz/TCO reaction, a diffusion-controlled interfacial crosslinking method was devised for the fabrication of a hydrogel sphere with a 3D core-shell pattern. By changing the ratio of the monofunctional capper and the bifunctional TCO crosslinker, hydrogels with a Young’s modulus ranging from 4 kPa to 20 kPa, comparable to that for human vocal fold LP, were obtained. Additionally, the diffusion-controlled method permitted precise spatial control of the enzymatic degradability and cell adhesivity in core and shell layers where homogeneously encapsulated human mesenchymal stem cells (hMSCs) exhibited different cell morphologies at each layer. ☐ Finally, a bioorthogonal hydrogel platform that recapitulates dynamic alterations in matrix properties during wound healing was engineered. The slow Tz/Nb was utilized to establish the covalent hydrogel networks for 3D cell encapsulation. The rapid Tz/TCO ligation enabled real time in situ modulation of the density of RGD ligands. The diffusion-controlled reaction also allowed the time-delayed introduction of RGD peptides to cell-laden hydrogel constructs, mimicking the progressive deposition of fibronectin during the early stage of wound healing. Transforming growth factor beta 1 (TGFβ1)-induced myofibroblast differentiation from primary vocal fold fibroblasts was observed only at a high RGD concentration, as evidenced by the development of alpha-smooth muscle actin (αSMA) positive F-actin stress fibers. The RGD-driven phenotype change was accompanied by enhanced cytokine secretions and matrix remodeling. ☐ Overall, I presented a powerful bioorthogonal platform for spatiotemporal presentation of biochemical and biomechanical signals to direct cellular responses and differentiation. This work represents the first step towards the development of a hydrogel-based cellular model of the human vocal fold lamina propria in both healthy and diseased states.
Lamina propria, Bioorthogonal hydrogel platform, Vocal fold, Scrylate, Tetrazine