Characterization of silk proteins from African wild silkworm cocoons and application of fibroin matrices as biomaterials

Abstract

Challenges in treating injuries, together with an increased need for repair of damaged tissues and organs, have made regenerative medicine a major research area today. Biomaterials such as silk fibroin (SF) have proven to be excellent tissue scaffolds possessing properties essential in tissue engineering such as biocompatibility, biodegradability and exceptional mechanical properties. SF nanofibres are especially attractive due to their large surface-to-volume ratio and high porosity which is beneficial in regenerative medicine. However, to design biomaterial scaffolds, chemical and physical properties of SF have to be sufficiently known. The thesis aims to contribute to knowledge by characterizing silk fibroin from the African wild silkworm species Gonometa rufobrunnae, Gonometa postica, Argema mimosae, Epiphora bahuniae and Anaphe panda. Moreover, the feasibility of producing nanofibrous biomaterial scaffolds from these fibroins is explored. The chemical composition of degummed fibres was investigated using Capillary electrophoresis whilst Infrared (IR) and Raman spectroscopic techniques were utilized to determine structural characteristics of the fibroin. In addition, thermal behaviour and mechanical properties of the fibroins were also investigated. Nanofibres were fabricated via electrospinning. The effects of solution concentration, voltage, polymer flow rate and tip to collector distance were studied to give optimum electrospinning conditions. IR spectroscopy was also utilized to observe the conformational structure of the degummed and electrospun fibres whilst scanning electron microscopy (SEM) provided information on the size and morphology of the fibres. The use of the nanofibres as biomaterials was evaluated using cytotoxicity tests. Results showed that glycine, alanine and serine constituted over 70% of the amino acid composition of all the fibroins. Gonometa fibroin had more glycine than alanine whilst the opposite was true for Argema mimosae, Epiphora bahuniae and Anaphe panda fibroin. The abundance of basic amino acids in Gonometa rufobrunnae, Gonometa postica, Argema mimosae and Epiphora bahuniae fibroin makes them prime candidates for cell and tissue culture. The amino acid composition of the fibroins influenced secondary structure as the β-sheet structure. Anaphe panda, Argema mimosae and Epiphora bahuniae silks was made up of mostly alanine-alanine (Ala-Ala)n polypeptides whilst Gonometa fibroin had an interesting mixture of both glycine-alanine (Gly-Ala)n and (Ala-Ala)n units. The unique structures impacted the mechanical and thermal properties of the fibroins. Production of Gonometa nanofibres was mainly dependent on fibroin solution concentration. A minimum of 27 % w/v was needed to produce defect free nanofibres. Diameters of the electrospun fibres produced ranged from 300 to 2500 nm. IR spectroscopy data highlighted that the β-sheet conformation of degummed fibroin was degraded during the formation of the nanofibres rendering them water soluble. It was however possible to regenerate the β-sheet structure in the nanofibres by exposing them to various solvents. Cytotoxicity tests using Sulforhodamine B (SRB) assay demonstrated that the nanofibres were not toxic to cells, a major prerequisite for use as a biomaterial. This thesis successfully provides useful data in an area that has been minimally explored. Results suggest that SF from African silkworm species offers diversity in properties and are therefore attractive for use as biomaterials, especially in cell and tissue engineering. As far as we could determine, we are the first to extend the use of fibroin from African silk species by producing Gonometa SF nanofibres that are of potential use as biomaterial scaffolds.