Freeze–thaw cycles for biocompatible, mechanically robust scaffolds of human hair keratins

The keratin-based scaffolds are getting more and more attention in the application of tissue engineering. Though various approaches have been considered to improve the physical properties of these scaffolds, few succeeded in achieving the enhanced properties of the pure keratin scaffolds. Due to the presence of -OH, -NH₂ , >CO, and -SH on the extracted human hair keratin (HHK), the formation of hydrogen bonds and disulfide bridges could be triggered under certain conditions, leading to the self-cross-linking of HHK materials. Herein, a simple and green strategy was introduced, via freeze-thaw cycles of keratin solutions without addition of extraneous reagents, to obtain the mechanically robust HHK scaffolds. The comparative quantitation of residual -SH among the samples treated with 1, 5, and 9 cycles confirmed the oxidation in the thaw process for forming disulfide bonds. So, the equivalent thaw time was applied in this study, and three groups of the treated samples after 1, 5, and 9 cycles with an appropriate extension thaw time were prepared to solely investigate the effects of physical cross-linking networks, primarily by formation of hydrogen bonds, on the properties of the obtained scaffolds. The systematic assessments including swelling behavior, porosity, thermal analysis, compressive measurement, and microstructural observation confirmed that the repetitive freeze-thaw treatment contributed to mechanically robust scaffolds with good porous interconnectivity. The cell culturing experiments further verified that these HHK scaffolds had desirable cytocompatibility, permitting the proper proliferation, attachment, and infiltration. Accordingly, this study provided a simple and efficient method to obtain biocompatible, mechanically robust keratin scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1452-1461, 2019.