Three-dimensional hMSC motility within peptide-functionalized PEG-based hydrogels of varying adhesivity and crosslinking density

Human mesenchymal stem cell (hMSC) migration and recruitment play a critical role during bone fracture healing. Within the complex three-dimensional (3-D) in vivo microenvironment, hMSC migration is regulated through a myriad of extracellular cues. Here, we use a thiol–ene photopolymerized hydrogel to recapitulate structural and bioactive inputs in a tunable manner to understand their role in regulating 3-D hMSC migration. Specifically, peptide-functionalized poly(ethylene glycol) hydrogels were used to encapsulate hMSC while varying the crosslinking density, from 0.18 ± 0.02 to 1.60 ± 0.04 mM, and the adhesive ligand density, from 0.001 to 1.0 mM. Using live-cell videomicroscopy, migratory cell paths were tracked and fitted to a Persistent Random Walk model. It was shown that hMSC migrating through the lowest crosslinking density and highest adhesivity had more sustained polarization, higher migrating speeds (17.6 ± 0.9 μm h−1) and higher cell spreading (elliptical form factor = 3.9 ± 0.2). However, manipulation of these material properties did not significantly affect migration persistence. Further, there was a monotonic increase in cell speed and spreading with increasing adhesivity that showed a lack of the biphasic trend seen in 2-D cell migration. Immunohistochemistry showed well-formed actin fibers and β1 integrin staining at the ends of stress fibers. This thiol–ene platform provides a highly tunable substrate to characterize 3-D hMSC migration that can be applied as an implantable cell carrier platform or for the recruitment of endogenous hMSC in vivo.