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

Abstract

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.

Dual‐chambered membrane bioreactor for coculture of stratified cell populations

“…The DCB (Figure c) provides two parallel flow lines into and out of a common chamber. The inclusion of a membrane limits mixing and convective flow between lines, and regulates diffusive transport depending on its crosslinking degree (CD; Navarro et al, ).…”

“…It should be stated that any porous membrane could be used for this purpose in the DCB. Here, we chose to implement keratin hydrogels (Figure a) for this purpose because we have developed a method to alter its crosslinking degree by regulating the amount of energy delivered to the sample during crosslinking (Navarro et al, ). This gave us the possibility to study regulation of communication in the bioreactor by changing the properties of the membrane, properties that we ourselves could design, control, and manufacture as described in our previous studies.…”

“…Keratin was extracted from oxidized human hair using a proprietary method by KeraNetics LLC (Winston‐Salem, NC; Placone et al, ; Tomblyn et al, ). Keratin‐based photocrosslinkable resin was prepared as previously reported (Navarro et al, ; Placone et al, ). Briefly, keratin was dissolved in PBS (pH 7.4) at a 4% wt/vol concentration, and then mixed with a photosensitive solution at a 4:1 ratio.…”

“…The resin was used in 3D printed molds to cast membranes (12 × 12 × 1.5 mm), exposing to UV at an intensity of 350 mW/dm 2 . Membranes were produced with 20% low and 59% high crosslinking degree (LCD and HCD) by regulating the amount of energy delivered to the sample during crosslinking (Navarro et al, ). Samples were thoroughly rinsed to remove unreacted components and stored in PBS at 4°C.…”

“…We previously reported the development of keratin‐based hydrogel membranes that can be fine‐tuned to regulate transport across them (Navarro, Swayambunathan, Lerman, Santoro, & Fisher, ; Navarro, Swayambunathan, Santoro, & Fisher, ). Here we assessed the viability of our DCB for growing adjacent cell populations with the inclusion of a keratin membrane.…”