Enzymatic Cross-Linking of Resilin-Based Proteins for Vascular Tissue Engineering Applications.

Protein-based biomaterials have received significant attention for tissue engineering applications. For example, resilin-based protein gels have been produced with different cross-linking chemistries for applications in cartilage, cardiovascular, and vocal fold engineering. In this study, we investigate an alternative cross-linking chemistry to form resilin-based protein hydrogels and demonstrate the versatility of the gels for investigating cell response to matrix stiffness. Specifically, transglutaminase was used to cross-link proteins and resulted in gel surfaces more suitable for long-term cell attachment compared to those formed by a Mannich-type condensation reaction. Since matrix stiffness is an important determinant in modulating cell response, we first tuned matrix stiffness by varying total protein concentration. Next, we observed that matrix stiffness modulated cell spreading and endothelial differentiation of human mesenchymal stem cells. In particular, our results show that cells differentiated on our matrices, which have a stiffness similar to subendothelial layers, had statistically equivalent endothelial function compared to cells differentiated on hard glass surfaces. Thus, our protein-based matrix system is a promising tool to provide substrates favorable for long-term cell attachment and better mimics the native subendothelial environment compared to conventional hard culture substrates.

Characterization of resilin-like proteins with tunable mechanical properties.

Resilin is an elastomeric protein abundant in insect cuticle. Its exceptional properties, which include high resilience and efficient energy storage, motivate its potential use in tissue engineering and drug delivery applications. Our lab has previously developed recombinant proteins based on the resilin-like sequence derived from Anopheles gambiae and demonstrated their promise as a scaffold for cartilage and vascular engineering. In this work, we describe a more thorough investigation of the physical properties of crosslinked resilin-like hydrogels. The resilin-like proteins rapidly form crosslinked hydrogels in physiological conditions. We also show that the mechanical properties of these resilin-like hydrogels can be modulated simply by varying the protein concentration or the stoichiometric ratio of crosslinker to crosslinking sites. Crosslinked resilin-like hydrogels were hydrophilic and had a high water content when swollen. In addition, these hydrogels exhibited moderate resilience values, which were comparable to those of common synthetic rubbers. Cryo-scanning electron microscopy showed that the crosslinked resilin-like hydrogels at 16 wt% featured a honeycomb-like structure. These studies thus demonstrate the potential to use recombinant resilin-like proteins in a wide variety of applications such as tissue engineering and drug delivery due to their tunable physical properties.