Abstract
Wool derived keratin has garnered significant advancements in the field of biomaterials for hard tissue regeneration. Keratin has a three-dimensional structure which hinders the dissolution in common solvents. Therefore, harsh conditions and toxic chemicals are required for their dissolution and processing. The main purposes of this thesis were twofold. The first aim was to extract keratin from sheep wool using a novel green approach based on ionic liquid solvents. The properties of the extracted keratin were studied by using various analytical techniques. The second aim was to fabricate a biomimetic dual layered keratin/hydroxyapatite scaffolds utilizing a freeze-drying technique. The chemical, structural, and physical properties of the developed keratin scaffolds were characterized. Furthermore, the biocompatibility of the characterized scaffolds was investigated using Saos-2 cell culture testing. The findings of this study demonstrated that the extraction technique employed was effective in isolating the intermediate filament proteins. FTIR analysis showed that the extracted keratin maintains the polypeptide backbone structure. SEM studies confirmed that the compact structure of keratin was lost after interaction with ionic liquid. TGA analysis showed that the thermal stability of the keratin was higher compared to raw wool. The composite dual gradient porous scaffolds were characterized in terms of their physiochemical properties. The FT-IR analysis confirmed that the secondary structure of keratin was not destroyed with the successful incorporation of hydroxyapatite particles during the process of scaffold fabrication. X-ray diffraction analysis showed that the keratin and hydroxyapatite retained their crystalline structures in the prepared keratin/HA scaffolds. SEM studies showed an interconnected porous architecture of the prepared scaffolds with seamless integration between the upper and lower layers. EDX analysis showed that the inorganic phases of both the upper and lower layers of the keratin/HA scaffold mainly consist of calcium and phosphorous ions. EDX mapping confirmed the even distribution of calcium (Ca) and phosphorous (P) in both the upper and lower layers of the scaffold. The prepared scaffolds showed a gradual degradation during the 28-day incubation period in PBS.The incorporation of HA improved the mechanical properties keratin/HA scaffolds. The keratin/HA scaffolds exhibited superior mechanical properties in terms of Young’s modulus and compressive strength in comparison to pure keratin scaffolds. The biocompatibility studies suggested that both keratin and keratin/HA scaffolds were cyto-compatible, in terms of cell viability and proliferation. Furthermore, it showed that both the tested materials can served as an ideal substrate for the differentiation of Saos-2 cells, leading to mineralization of the extracellular matrix. In summary, the development of a non-toxic green method was investigated for keratin extraction to fabricate keratin/HA scaffolds and our results showed great potential for the use of these scaffolds to regenerate alveolar bone due to their structural similarity and excellent in vitro biocompatibility.