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.