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Hydrogels are polymeric materials characterized as an organized cross-linked networks that absorb and retain substantial amounts of water. Hydrogels have been explored in several promising application areas such as wound healing, wound dressings and skin substitutes. Among macromolecules investigated for hydrogels fabrication, keratin shows interesting potential due to their abundant availability, improved cell adhesion properties, in vivo biocompatibility and accelerated wound-healing properties. Keratins are a family of intermediate filament proteins found in the structures of different tissues in mammals (hair, nails, skin, fur, sheep wool, horns, and hooves), and birds (e.g. bird feathers and beaks). However, mammalian and avian keratins show distinct variations among amino acid composition, molecular weight, and protein secondary structures. Current literature lacks comprehensive studies on the fabrication and characterization of keratin hydrogels from different sources while prepared under the same conditions. The objectives of this study were to characterize and compare the rheological (storage modulus), physical (porosity, pore size, swelling capacity, and water contact angle) and in vitro cell compatibility of hydrogel scaffolds prepared from mammalian and avian keratins.
In this work, keratins from human hair, sheep wool and chicken feathers were used to prepare hydrogel scaffolds by self-assembly of keratin solutions through dialysis. Keratins were extracted by sulfitolysis and characterized for their chemical and conformational properties (amino acid composition, molecular weight, secondary structure, and thermal properties). Male hair, Merino wool and white chicken feathers were obtained for keratin extraction. Keratin gels were prepared by re-solubilizing keratin powders at 7.5% w/v in KES at pH 7 for 4 h. at room temperature. After 5 days of dialysis gels were cut into 16 mm diameter discs, placed in 24-well culture plates, frozen and lyophilized. Dry gels were cut into specimens of 2 mm thickness and used as cellular scaffolds. Hair, wool and chicken feathers were digested with 6 N HCl for quantification of all amino acids, with exception of cysteine. For cysteine, separate samples were previously hydrolyzed with performic acid. Total nitrogen in keratinous samples was determined using a Leco Truspec C/N analyzer. Thermal properties of raw materials and extracted keratins were studied by differential scanning calorimetry (DSC) in a DSC Q2000 instrument.
Hair and wool keratins are heterogeneous proteins with molecular weights ranging from around 10 to 75 kDa. Feather keratins are smaller proteins of around 10 kDa. While hair and wool keratins are composed of around 400–500 amino acid residues, feather keratins are smaller proteins composed of around 100 amino acids residues. The highest proportion of non-polar/polar amino acids was determined in chicken feathers (51/49%) followed by wool (43/57%) and hair (34/66%). Hydrogels prepared from keratin sources showed different appearance. Feather keratins at 7.5% w/v formed firm gels that were easy to handle and cut into discs, while gels from hair and wool keratin were weaker and more difficult to handle. Keratin hydrogels were freeze-dried to form porous materials that could be used as dermal scaffolds and ultimately for tissue regeneration of skin injuries. Feather keratin scaffolds showed the largest mean pore size (209.5 μm).
The results of this study could help understand the gelation mechanisms of different keratins and the modulation of viscoelastic and microstructural properties for their application in tissue engineering. Smaller molecular weight and β-sheet conformation of feather keratins resulted in more mechanically robust hydrogels. Higher molecular weight and α-helix conformation of hair and wool keratins resulted into weaker, but flexible hydrogels able to absorb high amounts of water (3000%). Fibroblasts proliferated at higher rates in stiffer feather keratin hydrogel scaffolds. Partial degradation of feather keratin network was observed after 15 days of cell culture. Stronger hydrogels from avian keratins could serve in the fabrication of advanced wound healing biomaterials. Further in vitro and in vivo evaluation of hydrogel scaffolds would support the potential of feather keratins for would healing applications.