Development of keratin-based membranes for potential use in skin repair

The layers in skin determine its protective and hemostasis functions. This layered microstructure cannot be naturally regenerated after severe burns; we aim to reconstruct it using guided tissue regeneration (GTR). In GTR, a membrane is used to regulate tissue growth by stopping fast-proliferating cells and allowing slower cells to migrate and reconstruct specialized microstructures. Here, we proposed the use of keratin membranes crosslinked via dityrosine bonding. Variables from the crosslinking process were grouped within an energy density (ED) parameter to manufacture and evaluate the membranes. Sol fraction, spectrographs, and thermograms were used to quantify the non-linear relation between ED and the resulting crosslinking degree (CD). Mechanical and swelling properties increased until an ED threshold was reached; at higher ED, the CD and properties of the membranes remained invariable indicating that all possible dityrosine bonds were formed. Transport assays showed that the membranes allow molecular diffusion; low ED membranes retain solutes within their structure while the high ED samples allow higher transport rates indicating that uncrosslinked proteins can be responsible of reducing transport. This was confirmed with lower transport of adipogenic growth factors to stem cells when using low ED membranes; high ED samples resulted in increased production of intracellular lipids. Overall, we can engineer keratin membranes with specific CD, a valuable tool to tune microstructural and transport properties.