Développement de matériaux à base de protéines extraites du byssus de la moule bleue Mytilus edulis

The byssus is a set of protein-based anchoring threads produced by marine mussels to tether to water immersed surfaces. These fibers have impressive mechanical properties combining stiffness, elasticity and toughness, as well as a self-healing behavior of their mechanical performance upon rest following removal of stress. These properties are the result of collagen-rich block copolymer-like proteins hierarchically assembled and of the presence of organo-metallic reversible sacrificial bonds. Even though these fibers have outstanding mechanical properties and a high content of potentially biocompatible collagen, the mussel farming industry still treats them as a waste. The main objective of this thesis was to use byssus as a sustainable biological feedstock to produce a new family of biomimetic protein-based materials. We developed a method to produce a byssus protein hydrolyzate (BPH) rich in charged amino acids (~30 % mol) and with good film-forming capabilities. A hydrogel rich in inter- and intra-molecular anti-parallel β-sheets and in collagen triple helical structures forms following the BPH solubilization at pH 10.5. After evaporation of water, the resulting film is insoluble in aqueous media as a result of the BPH self-assembly into stable secondary structures. The mechanical properties of the films are pH-responsive owing to their high electrostatic charges content that act like effective crosslinking points at the isoelectric point (pI = 4.5), but causes swelling of the protein matrix and loss of mechanical performance at pH higher or lower than the pI. The strain at fracture remains constant, which increases the toughness of the materials when moving toward the pI. The high content in charged and reactive amino acids was used to covalently crosslink the BPH films using either EDC or glutaraldehyde. Increasing the crosslinking degree gives rise to stiffer and stronger films but leads to a loss of extensibility as a consequence of protein chains being trapped by the crosslinking points. The crosslinked films become resistant to collagenase degradation even though infrared spectroscopy shows the conversion of aggregated strands to hydrated collagen/PPII related structures following the crosslinking reaction. Thus, the crosslinked collagen-related structural elements hinder the collagenase action on the BPH films. Sacrificial bonds were formed in the BPH films by treatments at their pI and/or with various metallic ions, i.e. Na+, Ca2+, Fe3+, in order to tune the mechanical properties and to evaluate the role of sacrificial bonds on the self-healing behavior during cyclic mechanical testing. Using metallic ions of higher valence to treat the films results in an increase of the modulus, strength and toughness without reducing the strain at fracture, confirming the formation of organo-metallic sacrificial bonds. Cyclic mechanical testing shows that pI and Ca2+ treatments create reversible ionic sacrificial bonds that induce a pH-dependent self-healing behavior. Fe3+ addition at various concentrations enables tuning the mechanical performances over a larger interval and to reach higher values than other treatments. This behavior is attributed to the more covalent nature of the iron-amino acids bonding and to the affinity of iron with numerous amino acids, including histidines and DOPA, as detected using Raman and infrared spectroscopy. Iron addition also leads to the formation of reversible sacrificial bonds that procure a self-healing behavior of the mechanical properties to BPH films. Altogether, our results show that the BPH films are byssus biomimetic hydrogels whose mechanical properties can be tuned by using various treatments or crosslinking reactions. The materials could thus find a niche as protein matrix in domains such as the pharmaceutical industry or soft tissue engineering.

Keyphrases: Byssus, Proteins, Crosslinking, Spectroscopy, Mechanical properties, Sacrificial bonds, Biomimetism