A NEW KERATIN-BASED SCAFFOLD WITH PROMISING FEATURES FOR BIOMEDICAL APPLICATION

Introduction: Natural polymers exhibit several features that make them more biocompatible than the synthetic ones. One of the most studied natural polymers is the keratin, an abundant non-food protein found in hairs, wool and nails of mammals. It is highly hydrophilic and contains a high amount of cysteine, a sulphur-containing amino acid that gives rise to intra- and intermolecular disulphide cysteine bonds which largely influence its mechanical and chemical properties. Keratin can be extracted from natural sources using reducing or oxidizing agents, and successively regenerated in the forms of films, nanofibers, sponges and hydrogels. In this study, sheep's wool was used as a natural source to prepare keratin microfibril sponges for scaffolding, by disruption of the histological structure of the fibers through chemicalphysical treatment. Subsequently, biocompatibility of wool sponges was evaluated using the osteoblast-like SAOS-2 cell line. Materials and methods: Wool sponges were prepared using keratin extracted from sheep's wool throughout alkali treatment, followed by ultrasonication, casting and salt leaching. Morphological characterization was performed by analyzing sponge porosity and degree of crosslinking throughout SEM observations. Moreover sponge tensile and compression behaviors were studied using dynamometer according to the EN-ISO 5079 in dry and wet conditions. SAOS-2 cells were selected for biocompatibility-cell viability assay and represent an in vitro model for osteoblast studies. Cells were seeded on scaffolds and cultivated for 1, 3 and 7 days when MTT and FDA assays were performed to determine cell viability. Cell morphology was evaluated through SEM observations and actin and tubulin filaments were stained to study cell adhesion on keratin scaffolds. Results: The wool sponges showed highly interconnected porosity (93%) and contain intrinsic sites of cellular recognition that mimic the extracellular matrix. Sponges were stable in water without structural changes and showed excellent resilience to repeated compression stresses. According to in vitro biocompatibility assays, wool fibril sponges showed a good cell adhesion and proliferation. In fact, consistent cell proliferation (p<0.05) was reported for longer incubation times, showing a 2-fold and 4-fold increase related to the number of seeded cells after 3 and 7 days, respectively. After 24h, cells were widely spread on the wool fibril sponges as confirmed by SEM and confocal observations. At day 7, the wool fibril sponges were completely coated by cells and were even found in the pores. Discussion: The unique structure of the cortical cell network made by wool keratin proteins with controlled-size macro-porosity make this scaffold suitable for cell guesting and nutrient feeding. Both the compression traces of the dry and wet sponges display a horizontal line most likely due to reversible crushing deformation of the macropore structure. However, the wet sponges are more resilient because the compression traces are almost overlapping each other and no permanent deformation was detected. In summary, the wool fibril sponges made of keratin contain cellular-binding motifs that mimic the sites of cell attachment found in the native extra-cellular matrix components which facilitate better growth via providing proliferation signals to the cells and minimize apoptotic cell death.