Wool-derived keratin hydrogel as a potential scaffold for pulp-dentine regeneration: an in vitro and in vivo study

Abstract

Millions of people suffer from caries, which affect the quality of life of patients of all ages and result in an economic burden on healthcare systems worldwide. The current therapy is to remove the diseased tissues and to replace them with inert, synthetic materials that are incapable of replacing the biological function of the lost tissue, leading to tooth loss. This issue raises the need for biological treatment options which are able to maintain and renew tooth vitality. Similar to other connective tissue, pulp tissue has the potential to heal. Characteristics of the healing of exposed pulp tissue include reorganization of damaged soft tissue, differentiation of the dentine forming cells and tissue repair/regeneration. To achieve successful pulpo-dentinal regeneration, a highly porous scaffold that mimics the natural extracellular micro-environment is vital. Different natural and synthetic polymers have been investigated for this purpose. Of the naturally-derived biomaterials, keratin-based materials have shown promise for revolutionizing the biomaterial world due to their intrinsic ability to self-assemble; also, their biocompatibility, biodegradability, mechanical durability, and natural abundance. The aim of this thesis is to fabricate, characterize keratin hydrogel (KH) and assess its suitability for pulp-dentine regeneration. Keratin was extracted from sheep wool using a well-established extraction technique based on reductive chemistry. The extracted proteins were purified by dialysis, quantified by gel electrophoresis, mass spectrometry, amino acid analysis and inductively coupled mass spectrometry. The effect of keratin on the proliferation and differentiation of odontoblast-like cells and dental pulp stem cells was assessed by cell culture techniques. Analysis of cell viability, proliferation, differentiation and mineralization were carried out to assess their bioactivity. This was followed by fabrication of keratin hydrogels and their characterization based on structural, rheological and cell viability evaluations. Furthermore, the characterized KHs were investigated for their biocompatibility by implanting of the gel into exposed rat dental pulp. The subsequent reparative/regenerative pulpal response was assessed by histological and immunohistochemical analysis. The finding of this study demonstrated that the extraction technique employed was effective in isolating the intermediate filament proteins and these were predominantly Type I alpha keratins with ~7.5% cysteine content. The cell culture experiments to assess bioactivity demonstrated that 0.1mg/mL was the optimal keratin concentration required to enhance the proliferation and differentiation of dental pulp stem cells and odontotoblast-like cells. The characterized hydrogel was injectable with a highly porous microstructure that underwent slow degradation and had good cytocompatibility. The results of the animal study indicated that KH was biocompatible and able to form reparative dentine–like deposits after 28 d of implantation in vital dental pulp tissue. In summary, the isolated wool derived keratin intermediate filaments were cytocompatible, enhanced odontogenic differentiation behavior, biocompatible with reparative dentine formation and therefore, may provide an alternative biomaterial source for pulp-tissue engineering. The findings of this study may contribute in developing an alternate biological treatment option to the existing treatment modalities especially for pulp therapies.