Regulating cell phenotype and function using custom-designed synthetic matrices

Abstract

The natural extracellular matrix serves as a mechanical platform for cells and influences basic cellular functions. Using custom-designed, synthetic matrices, I investigated how matrix composition and properties affect cell differentiation, tissue remodeling and regeneration. ☐ In the first project, I investigated how the diameter of fibrous scaffolds affects epithelial-to-mesenchymal transition (EMT). I produced poly (b-caprolactone) (PCL)-based fibrous scaffolds with an average fiber diameter of 0.5 or 5.0 um. I discovered that Madin-Darby Canine Kidney cells grown on 0.5-um scaffolds adopted an epithelial phenotype and underwent EMT in the presence of a soluble EMT inducer. Contrarily, cells maintained on 5.0-um substrates were more mesenchymal-like and did not respond to the EMT inducer. Therefore, fiber diameter is a potent regulator of cell phenotype. ☐ In the second project, I investigated how matrix composition affects fibrogenesis. Fibrous scaffolds with a stiff PCL core (diameter: 3.8 um) and a soft, hyaluronic acid (HA) hydrogel shell (thickness: 0.1 or 0.7 um) were produced. I found that primary porcine vocal fold fibroblasts (PVFFs) cultured on fibers with a thin HA shell adopted a myofibroblast phenotype, whereas those maintained on fibers with a thick HA coating remained fibroblastic. Stimulation with a profibrotic growth factor, transforming growth factor β (TGFβ), promoted myofibroblast differentiation of cells cultured on both substrates. However, inhibition of Rho-associated kinases significantly attenuated the expression of fibrotic markers by cells grown on fibers with 0.7-um HA shell, but not the 0.1-um counterpart. Thus, PVFFs sense the thick HA shell and shift away from myofibroblast differentiation even in the presence of a soluble TGFb. ☐ The goal of the third project is to engineer a physiologically relevant vocal fold cover. HA-based synthetic matrices of varying composition and stiffness were produced. I discovered that primary porcine vocal fold epithelial cells (PVFEs) cultured on gels with an elastic modulus of 2,000 Pa and presenting laminin 111-derived AG73 peptide matured into a stratified epithelium, consisting of a basal, proliferating cells, and upper, differentiated layers. Identifying matrix properties required for PVFE differentiation and stratification is a first step toward an engineered in vitro model of the human vocal fold. ☐ In the final project, I investigated how distinct peptide signals mediate the differentiation of human salivary/stem progenitor cells (hS/PCs). HA-based hydrogels with covalently conjugated peptide derived from fibronectin (RGDSP), perlecan domain IV (TWSKV), and laminin 111 (YIGSR and IKVAV) were synthesized. I found that RGDSP and IKVAV signals promoted hS/PC differentiation to the ductal lineage, whereas TWSKV and YIGSR signals induced acinar differentiation. Identifying peptide ligands that induce hS/PC commitment to specific cell types found in the native gland helps pave the way for the development of an engineered salivary gland. ☐ Overall this is work highlights the importance of biochemical, topographical and biomechanical signals of the extracellular matrix in regulating cellular interactions and fostering cell differentiation.