Designed Protein-Based Physical Crosslinker for Triple Helical Collagen Biomaterials

Abstract

Collagen plays a pivotal role in animal body structure, but hydrogels of reconstituted collagens often have poor mechanical performance. Chemical crosslinking or the addition of other biopolymers are often used as a remedy. Inspired by the many natural collagen-interacting proteins that modify the mechanics of collagen hydrogels, we explore protein-based physical crosslinking of collagen as a route to modulate the mechanics of collagen hydrogels. A protein-based crosslinker for collagen was designed based on an adhesion domain from Staphylococcus aureus. A tetramerization domain was coupled to an elastin-like polypeptide as a spacer, followed by the collagen adhesion domain, yielding a tetrafunctional crosslinking. Circular Dichroism spectroscopy shows that the crosslinker has the expected secondary structure. Using quartz crystal microbalance, we demonstrate the tetrameric crosslinker binds rat tail Collagen I and fish collagen, even though the latter is largely denatured as evidenced by circular dichroism spectroscopy. Rheometry shows strong enhancement of elastic moduli and strain-stiffening for rat tail collagen hydrogels, but smaller effects on the reduced fish collagen. Because CNA35 recognizes the collagen triple helix, this contrast indicates that the crosslinker acts specifically on triple-helical collagen, and points to the importance of improved extraction protocols for alternative collagens that preserve native triple-helical conformations.