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Recombinant Collagen Variants for the Production of Mechanically and Biofunctionally Tunable Hydrogels

  • Author(s): Que, Richard Alan
  • Advisor(s): Wang, Szu-Wen
  • Da Silva, Nancy A
  • et al.
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License
Abstract

As the most abundant protein in humans, collagen has been utilized as a tissue engineering scaffold. However, it exhibits the same limitations as other natural substrates, such as the inability to decouple the modulation of mechanical and biofunctional properties. The properties of the cell microenvironment guide cell fate, making it advantageous to modulate the properties of the microenvironment. In this work, we employed a modular collagen platform to produce recombinant collagen variants with prescribed functionalities to independently tune the properties of collagen. Collagen variants with two to eight non-native cysteines were synthesized to form hydrogels of varying stiffness while holding protein and functional site concentration constant. The variants were characterized by circular dichroism and found to be triple-helical with melting temperatures near 37°C. Hydrogel gelation characteristics and stiffness were measured through microrheology and bulk rheology. Non-native cysteines were also utilized as specific covalent anchoring sites for TGF-β, which induced myofibroblast differentiation of fibroblasts that were cultured on these substrates.

We also manipulated integrin-binding sites, which are known to affect cell fate. Using a collagen variant with its integrin-binding sites removed, we tested the effects of the α2β1 integrin-binding sequence GFOGER from collagen I. By introducing GFOGER at up to four locations throughout the polymer, we demonstrated that the location at which GFOGER was introduced affected cellular adhesion. One location resulted in 39% of the cells adhered compared to native collagen III, while other locations along the polymer restored native levels of cellular adhesion. This variation hints at the importance of local context on the functionality of the inserted bioactive sequences and that additional modulation of properties can be achieved by placing the functional sequences at different locations throughout the polymer. Furthermore, modulation of cellular adhesion levels was achieved through mixing collagen substrates at different ratios.

We also demonstrated that the modular collagen platform also allows for the mixing-and-matching of fragments with distinct functionalities. Non-native cysteines and GFOGER were introduced within the same collagen protein to test the effect of GFOGER sites in 3D culture. NHLFs were successfully encapsulated within collagen-variant hydrogels. NHLFs remained spherical when encapsulated with a non-cell adhesion supporting collagen variant, while introduction of GFOGER into this variant resulted in NHLFs invading the gel. This research demonstrates the ability to produce full-length collagen variants with customizable functionalities to tune hydrogel stiffness and integrin-binding site presentation for potential tissue engineering applications such as vascularization.

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