Dermal fibroblasts with impaired migration for in vitro models of aged wound healing for testing new biomaterials-based therapies for chronic wounds

摘要

Introduction: Healthy wound healing involves inflammation, new tissue formation and finally remodelling as orchestrated stages leading to tissue repair. Ageing significantly affects wound healing in the geriatric population, more prone to chronic wounds, which in the UK alone cost ￡4 billion annually. In vitro models that mimic the in vivo scenario would be advantageous to test new biomaterial-based therapies for addressing the clinically challenging issue of chronic wounds. New dermal biomaterials developed for treating chronic wounds should be tested under aged wound healing conditions to better understand and predict how they would behave in vivo. For this purpose, a population of aged dermal fibroblasts, which attach normally but show deficits in migration due to a significant reduction in α2β1 integrin function, could be generated: when the new dermal biomaterial is implanted it will be populated by aged dermal fibroblasts, the main cell type in the dermis. Materials and Methods: Synthetic Arg-Gly-Asp (RGD) pepticles, which bind to integrins, and cost-effective, simple techniques and equipment were used to generate primary normal human dermal fibroblasts (pnHDFs) that mimic the behaviour of aged dermal fibroblasts in vivo. A scan of conditions was tested using an alamarBlue~? assay on two different surfaces of proteins commonly used in the development of biomaterials for tissue repair and which contain RGD sites (fibrinogen and gelatin) as well as on polystyrene (RGD sites free surface). Conditions scanned were: RGD pepticles concentration (0-10.67mM), +/- pre-incubation of cells with RGD pepticles for 30 min at 37°C with 5% CO2 and incubation time of cells at 37°C with 5% CO2 (2,24 or 48 h). Phase-contrast light microscopy (cell morphology) and a trypan blue assay (viability and cell number) on both attached and unattached cells and a series of scratch assays (cell migration) were performed on the chosen conditions. Finally, immunocytochemistry followed by confocal microscopy was done to study integrin expression and migration. Results and Discussion: Results showed that attachment and migration of pnHDFs can be tuned with synthetic RGD pepticles without affecting integrin expression and cell viability. In our study a concentration of 2.67mM of RGD pepticles impaired migration without significantly affecting cell attachment and integrin expression, thus mimicking the behaviour of aged dermal fibroblasts in vivo. Results suggest that the components of the migration pathways are intact but migration is impaired due to integrins binding to the RGD pepticles used in this study. Finally, we found that the number of RGD sites of the surface modulates the effect of the RGD pepticles on cell attachment and migration. Conclusion: Attachment and migration of pnHDFs can be tuned with synthetic RGD pepticles without affecting integrin expression and cell viability. These cells could be used in in vitro models of aged wound healing to test new biomaterial-based therapies for treating chronic wounds. We are currently using RGD-treated pnHDFs to study attachment and migration in 3D dermal scaffolds, thus testing them in an in vitro environment that mimics the in vivo scenario.