Keratin Extraction, Iso-Electric Precipitation and Micro-Pattern Preparation for Cellular Contact Guidance

The waste stream of low-grade wool from the fabric preparation is a keratin-rich material currently underutilized. Therefore, development of appropriate waste employment for added value final products is required. Wool is composed of 95% amount of keratin proteins by weight that have 5-10 mol% cysteine residues. The presence of intra- and intermolecular bonding, including the disulfide bridges formed between cysteine residues, ionic bonds, hydrogen bonds or hydrophobic bonds make the structure of keratin strong but, conversely, difficult to extract and reprocess. Consequently, key to successful keratin extraction is the selection of appropriate chemicals to break these bonds [1]. Moreover, keratin chains contain the sequence leucine-aspartic acid-valine, a well-established cell adhesion motif: this tripeptide is recognized by the cell adhesion molecule α₄β₁ integrin. α₄β₁ integrin can be found in several cells [2]; more specifically, the β₁ integrin subunit plays a crucial role as mechano-sensory receptor in dermal fibroblasts, regulating tissue homeostasis and skin wound healing. This may imply that keratin can potentially act as a tissue regeneration template. Therefore, preparation of keratin micropatterns could become an appropriate strategy for directing cell growth and tissue regeneration. In the present study, we would like to investigate the efficiency of keratin extraction with the high yield target and to explore the possibilities of keratin particles preparation using isoelectric precipitation (IEP). Furthermore, we would like to utilize micro-contact printing (MCP) technique to fabricate keratin-based micropatterns and evaluate their role in the cellular guidance <i>in vitro</i>.<br/>The keratins were extracted from wool using thermo-chemical treatment, such as sulfitolysis, hydrolysis and reduction [3]. These methods were selected due to their high efficiency reported in the literature. Keratin particles were prepared using IEP. The zeta potential as a function of pH, isoelectric point (IP), morphological structures, molecular weights, chemical composition of precipitated keratins and the influence of an anionic surfactant on biocompatibility were investigated. Subsequently, the keratin-based micropatterns were prepared via MCP. Primary human dermal fibroblasts adult cells were plated onto the keratin-patterned surface and cell viability, morphology and adhesion were evaluated by means of immunostaining and the confocal microscope imaging.<br/>The keratins prepared with sulfitolysis and reduction with cysteine showed good compromise between the molecular weights (both protocols produced keratins in the range of 10-60 kDa) and yield (63% and 56%, respectively). Moreover, these methods are based on non-toxic reagents; therefore keratins prepared using these protocols were used in the IEP. The zeta potential values of the surfactant-containing keratins were high and negative at all pH values while the surfactant-free samples showed a typical sigmoidal changes from negative to positive with decreasing pH and a neutral point corresponding to the isoelectric point (the point of minimum solubility). A thick layer of keratin precipitate that settled down at the IP was collected, washed and freeze-dried. IEP produced the globular, tightly packed nano- and microparticles and randomly arranged structures. Characterization techniques indicated that the chemical structure of proteins is retained after treatments. The traces of surfactant that were present in the keratins caused cytotoxicity of the samples, therefore only the surfactant-free samples were used for the keratin-based micro-patterns preparation. Keratin micro-stripes were found supportive in the facilitation of the specific cell adhesion and orientation.<br/>[1] A. Shavandi, et al. <i>Biomaterials science</i>, 2017, 5(9): p. 1699-1735.<br/>[2] V., S. Singh, et al. <i>Comprehensive Biomaterials II</i>, 2017, p. 542-557.<br/>[3] I. Sinkiewicz, et al. <i>Waste and biomass valorization</i>, 2017, 8(4): p. 1043-1048.