Nanoscale Molecular Quantification of Stem Cell−Hydrogel Interactions

A common approach to tailoring synthetic
hydrogels for regenerative medicine applications involves
incorporating RGD cell adhesion peptides, yet assessing the
cellular response to engineered microenvironments at the
nanoscale remains challenging. To date, no study has
demonstrated how RGD concentration in hydrogels affects
the presentation of individual cell surface receptors. Here we
studied the interaction between human mesenchymal stem cells
(hMSCs) and RGD-functionalized poly(ethylene glycol) hydrogels, by correlating macro- and nanoscale single-cell interfacial
quantification techniques. We quantified RGD unbinding forces
on a synthetic hydrogel using single cell atomic force
spectroscopy, revealing that short-term binding of hMSCs was
sensitive to RGD concentration. We also performed direct stochastic optical reconstruction microscopy (dSTORM) to
quantify the molecular interactions between integrin α5β1 and a biomaterial, unexpectedly revealing that increased integrin
clustering at the hydrogel-cell interface correlated with fewer available RGD binding sites. Our complementary, quantitative
approach uncovered mechanistic insights into specific stem cell-hydrogel interactions, where dSTORM provides nanoscale
sensitivity to RGD-dependent differences in cell surface localization of integrin α5β1. Our findings reveal that it is possible to
precisely determine how peptide-functionalized hydrog