Hydrogel-Supported, Engineered Model of Vocal Fold Epithelium

Abstract

There is a critical need for the establishment of an engineered model of the vocal fold epithelium that can be used to gain understanding of its role in vocal fold health, disease, and facilitate the development of new treatment options. Toward this goal, we isolated primary vocal fold epithelial cells (VFECs) from healthy porcine larynxes and used them within passage 3. Culture-expanded VFECs expressed the suprabasal epithelial marker cytokeratin 13 and intercellular junctional proteins occludin, E-cadherin, and zonula occludens-1. To establish the engineered model, we cultured VFECs on a hyaluronic acid-derived synthetic basement membrane displaying fibronectin-derived integrin-binding peptide (RGDSP) and/or laminin 111-derived syndecan-binding peptide AG73 (RKRLQVQLSIRT). Our results show that matrix stiffness and composition cooperatively regulate the adhesion, proliferation, and stratification of VFECs. Cells cultured on hydrogels with physiological stiffness (elastic shear modulus, G′ = 1828 Pa) adopted a cobblestone morphology with close cell–cell contacts, whereas those on softer matrices (G′ = 41 Pa) were spindle shaped with extensive intracellular stress fibers. The development of stratified epithelium with proliferating basal cells and additional (1–2) suprabasal layers requires the presence of both RGDSP and AG73 peptide signals. Supplementation of cytokines produced by vimentin positive primary porcine vocal fold fibroblasts in the VFEC culture led to the establishment of 4–5 distinct cell layers. The engineered vocal fold epithelium resembled native tissue morphologically; expressed cytokeratin 13, mucin 1, and tight/adherens junction markers; and secreted basement membrane proteins collagen IV and laminin 5. Collectively, our results demonstrate that stiffness matching, cell–matrix engagement, and paracrine signaling cooperatively contribute to the stratification of VFECs. The engineered epithelium can be used as a versatile tool for investigations of genetic and molecular mechanisms in vocal fold health and disease.

RGDSP-Decorated Hyaluronate Hydrogels Facilitate Rapid 3D Expansion of Amylase-Expressing Salivary Gland Progenitor Cells

“…Hydrogels were prepared by dissolving HA-SH in the peptide stock solution, followed by the addition of a pH-neutralized HA-AES solution. Under these conditions, peptide conjugation occurred through the rapid thiol–maleimide reaction, while the covalent network was established through the slower thiol–acrylate reaction , (Figure ). Peptide incorporation was quantified by analyzing the supernatant from swollen hydrogels with HPLC and comparing the chromatographs to standard curves constructed using unconjugated peptides (Figures S11–S16).…”

“…Determining the extent to which hS/PCs interact with the matrix when mechanically confined in a 3D network is nontrivial. 2D cultures are commonly performed to assess cell adhesion and to project cell–matrix interactions in 3D. , Thus, hS/PC affinity for each peptide was assessed by seeding hS/PCs on a stiffer (∼1800 Pa) gel to promote cell adhesion. Each hydrogel was prepared with 1 mM of peptide, and analysis was made by comparing with the number of cells on the blank HA gel (Figure A).…”

“…Thiol- and acrylate-functionalized HA derivatives, as HA-SH and HA-AES, respectively, were prepared according to our previously reported procedures. , In brief, sodium hyaluronate (430 kDa, Sanofi-Genzyme Corporation, Cambridge, MA) was dissolved in deionized water and reacted with 3,3′dithiobispropanoic dihydrazide in the presence of N -ethyl- N ′-(3-dimethylaminopropyl)­carbodiimide hydrochloride at pH 4.75. The reaction mixture was treated with 1,4-dithiothreitol, purified by dialysis, and lyophilized to yield HA-SH with 60% thiol incorporation, as confirmed by 1 H NMR.…”

A TGFβR inhibitor represses keratin-7 expression in 3D cultures of human salivary gland progenitor cells

“…Thiolated and acrylated HA derivatives (HA-SH and HA-AES) and maleimide-functionalized RGDSP (MI-RGDSP) were synthesized as previously reported 46 , 47 , 96 , 97 .…”

Editorial: Special Issue on Advanced Biomedical Hydrogels

“…Beyond the described fundamental understanding of hydrogel synthesis and control over processing, there are numerous specific applications of biomedical hydrogels that are emerging, including artificial tissue fabrication and controlled drug delivery systems. As an example of tissue models, engineered vocal fold epithelium is reported by Jia and colleagues . They developed hyaluronic acid-based vocal fold epithelium models to understand the role of epithelial cells in vocal fold health and disease.…”