(42g) Recombinant Collagen Variants for the Production of Tunable Hydrogel Scaffolds

As the most abundant protein in the human body, collagen has been a target for use as a tissue engineering scaffold. However, collagen from native sources suffers the same drawbacks as other natural substrates, including limitations in tuning of mechanical and biofunctional properties. Utilizing a novel biological synthesis method to address these challenges, recombinant human collagen variants containing 2, 4, and 8 non-native cysteines have been produced de novo.  As cysteines do not naturally occur within the triple-helical region of collagen, these sites provide specifically-defined locations for crosslinking or immobilization of bioactive factors, allowing for the tuning of the mechanical and signaling properties. Genes were synthesized using site-directed mutagenesis coupled with gene assembly, and the resulting biopolymers were expressed in an engineered Saccharomyces cerevisiae system.   For the purified recombinant collagen containing four non-native cysteines, triple-helical structure was confirmed by circular dichroism (CD), and melting temperature was measured to be near physiological temperature.  The recombinant collagen also possesses the ability to support cellular adhesion of HT-1080 human fibrosarcoma cells in vitro. Introduced cysteines were shown to be accessible and functional by conjugation with fluorophores.  By crosslinking these non-native cysteines, hydrogels were formed and gelation was monitored by particle tracking microrheology.  These collagen variants with introduced cysteines may be useful in the production of tunable hydrogels for tissue engineering applications with stiffness decoupled from binding, signaling, and degradation site densities. Additional functionality can also be introduced by utilizing non-native cysteines for the immobilization of factors that can augment tissue regeneration or guide cell fate.