Abstract
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 hydrogels interact with cells at the molecular scale, thus providing a basis to fine-tune the spatial presentation of bioactive ligands.
Highlights
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
We investigated how presenting a controlled amount of celladhesive peptide within a versatile hydrogel platform affected interfaced human mesenchymal stem cells from the macro- to nanoscale. This was accomplished by observing the migration speed of individual cells on a 2D hydrogel surface, measuring overall cell-hydrogel adhesions and individual rupture forces of cell-peptide interactions using single cell force spectroscopy (SCFS), and visualizing individual presentation and induced clustering of integrins on the cell membrane interfaced with the hydrogel using direct stochastic optical reconstruction microscopy
Our direct stochastic optical reconstruction microscopy (dSTORM) results fully support this increase in measured human mesenchymal stem cells (hMSCs) migration velocity on low binding RGD hydrogels through increased integrin surface localizations and clustering, suggesting that ligand density partly determines hMSC adhesion stability and migratory behavior
Summary
To explore the correlative relationship between the multiscale interfacing measurements, we indexed the various parameters between 0 and 1 (Figure 5). Even more precise exploration of how peptide-functionalized hydrogels interact with cells at the molecular scale appears to be in reach and would establish a basis for a priori determination of peptide amount, as well as its spatial and temporal presentation This setup is envisaged to be straightforward for use by many others as AFM is often used for material characterization, in addition to recent advances in single molecule interaction studies, the superresolution technique dSTORM, which can be carried out on a standard widefield microscope with the addition of a powerful laser and careful sample preparation. For the SCFS measurements, the hMSC-functionalized cantilever was positioned over a cell-free region of the hydrogel and force− distance curves performed.
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