Functionalized Biomimetic Hydrogels Enhance Salivary Stem/Progenitor Cell Organization

Complex branched salivary structures remain challenging to replicate within implant ready hydrogels. We showed previously that hyaluronic acid (HA)-based hydrogels enable growth and organization of primary salivary-derived human stem/progenitor cells (hS/PCs) into multicellular spheroids. Here, we systematically functionalized three components of migration-permissive hydrogels to foster salivary tissue morphogenesis. We separately analyzed contributions of an enzymatically degradable crosslinker, a pendant integrin-binding site, and hydrogel porosity to best support high viability, integrin-dependent cell adhesion and migration. Structure size, frequency, and morphology were all affected by hydrogel crosslink density and integration of biofunctional peptides. Viability and proliferation data suggested that integration of integrin binding sites had the greatest effect on hS/PCs behavior. A larger internal matrix space, created by increasing both crosslinker length and PEG content, was needed to form large multicellular hS/PC structures. Peptide-modified hydrogels with more internal space shifted hS/PC organization from spheroidal, surrounded by thick basement membrane, to an asymmetric arrangement with punctate matrix proteins defining a "wrinkled" perimeter. Integrin-binding peptides activated integrin {beta}1, with highest activation observed in hydrogels having both cleavable peptide and integrin ligand. The design parameters we prescribe allowed us to encapsulate hS/PCs in a humanized biomimetic hydrogel matrix able to support morphogenesis needed for salivary restoration.