Abstract
Collective processes such as wound re-epithelialization result from the integration of individual cellular decisions. To determine which individual cell behaviors represent the most promising targets to engineer re-epithelialization, we examined collective and individual responses of HaCaT keratinocytes seeded upon polyacrylamide gels of three stiffnesses (1, 30, and 100 kPa) and treated with a range of epidermal growth factor (EGF) doses. Wound closure was found to increase with substrate stiffness, but was responsive to EGF treatment only above a stiffness threshold. Individual cell behaviors were used to create a partial least squares regression model to predict the hierarchy of factors driving wound closure. Unexpectedly, cell area and persistence were found to have the strongest correlation to the observed differences in wound closure. Meanwhile, the model predicted a relatively weak correlation between wound closure with proliferation, and the unexpectedly minor input from proliferation was successfully tested with inhibition by aphidicolin. Combined, these results suggest that the poor clinical results for growth factor-based therapies for chronic wounds may result from a disconnect between the individual cellular behaviors targeted in these approaches and the resulting collective response. Additionally, the stiffness-dependency of EGF sensitivity suggests that therapies matched to microenvironmental characteristics will be more efficacious.
Highlights
The integration of individual cellular decisions to result in collective migration of keratinocytes is critical for wound re-epithelialization
In order to determine which of these individual cellular behaviors may represent potential future targets for therapeutic intervention, we characterized how combinations of EGF dose and substrate stiffness impacted keratinocyte collective migration and utilized this information to predict how changes in individual cellular decisions impact this collective response
Cell proliferation in the bulk and edge were differentially affected by substrate stiffness and EGF dose (Fig. 3), with bulk proliferation more responsive to EGF dose and edge proliferation more responsive to substrate mechanics on day 4 of wound closure
Summary
To determine if the different individual cellular processes involved in collective migration are impacted by differences in stiffness and EGF, we first quantified speed and persistence for single cells located at the wound edge. A stiffness-dependent effect was observed only when cells were treated with 10 ng/mL EGF; similar to the wound closure results for this condition (Fig. 1), cells migrated significantly faster and with significantly higher persistence on 100 kPa gels than on 30 kPa gels (p < 0.05).
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