Cartilage-like protein hydrogels engineered via entanglement.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.

Engineering shape memory and morphing protein hydrogels based on protein unfolding and folding.

Engineering shape memory/morphing materials have achieved considerable progress in polymer-based systems with broad potential applications. However, engineering protein-based shape memory/morphing materials remains challenging and under-explored. Here we report the design of a bilayer protein-based shape memory/morphing hydrogel based on protein folding-unfolding mechanism. We fabricate the protein-bilayer structure using two tandem modular elastomeric proteins (GB1)8 and (FL)8. Both protein layers display distinct denaturant-dependent swelling profiles and Young's moduli. Due to such protein unfolding-folding induced changes in swelling, the bilayer hydrogels display highly tunable and reversible bidirectional bending deformation depending upon the denaturant concentration and layer geometry. Based on these programmable and reversible bending behaviors, we further utilize the protein-bilayer structure as hinge to realize one-dimensional to two-dimensional and two-dimensional to three-dimensional folding transformations of patterned hydrogels. The present work will offer new inspirations for the design and fabrication of novel shape morphing materials.

Decorating protein hydrogels reversibly enables dynamic presentation and release of functional protein ligands on protein hydrogels.

Utilizing protein fragment reconstitution, we demonstrate the reversible and repeatable functionalization of protein hydrogels. This novel method enables the presentation and release of functional protein ligands on protein hydrogels.

Dynamic protein hydrogels with reversibly tunable stiffness regulate human lung fibroblast spreading reversibly.

We report the engineering of a protein-based dynamic hydrogel that can be reversibly tuned between stiff and soft states via a redox reaction. When cultured on this hydrogel surface, human lung fibroblasts can dynamically and reversibly change their morphology in response to changes in hydrogel stiffness.

Optically controlled reversible protein hydrogels based on photoswitchable fluorescent protein Dronpa.

Exploiting the optically controlled association and dissociation behavior of a photoswitchable fluorescent protein, Dronpa145N, here we demonstrate the engineering of an optically switchable reversible protein hydrogel using Dronpa145N-based protein building blocks. Our results open the possibility to optically tune the mechanical, chemical and structural properties of protein hydrogels.

Metal Chelation Dynamically Regulates the Mechanical Properties of Engineered Protein Hydrogels.

Engineering protein hydrogels with dynamically tunable mechanical and physical properties is of great interest due to their potential applications in biomedical engineering and mechanobiology. In our recent work, we engineered a novel dynamic protein hydrogel using a redox responsive, mutually exclusive protein (MEP)-based folding switch as the building block. By modulating the redox potential, the MEP-based folding switch can switch its conformation between two distinct states, leading to a significant change of the proteins' effective contour length of the polypeptide chain and an effective change of the cross-linking density of the hydrogel network (Kong, N. et al. Adv. Funct. Mater. 2014, 24, 7310). Building upon this work, here we report an engineered metal-chelation based method to dynamically regulate mechanical and physical properties of MEP-based protein hydrogels. We engineered a bihistidine metal binding motif in the host domain of the MEP. The binding of bivalent ions (such as Ni2+) enhances the thermodynamic stability of the host domain and results in the shift of the conformational equilibrium between the two mutually exclusive conformations of the MEP. Thus, the bihistidine mutant can serve as a metal ion responsive-folding switch to regulate the conformational equilibrium of the MEP. Using this bihistidine mutant of MEP as building blocks, we engineered chemically cross-linked protein hydrogels. We found that the mechanical and physical properties (including Young's modulus, resilience, and swelling degree) of this hydrogel can be regulated by metal chelation in a continuous and reversible fashion. This dynamic change is due to the metal chelation-induced shift of the conformational equilibrium of the MEP and consequently the effective cross-linking density of the hydrogel. Our results demonstrate a general strategy to engineer MEP-based dynamic protein hydrogels that may find applications in mechanobiology and tissue engineering.

Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry.

Constructing hydrogels from engineered proteins has attracted significant attention within the material sciences, owing to their myriad potential applications in biomedical engineering. Developing efficient methods to cross-link tailored protein building blocks into hydrogels with desirable mechanical, physical, and functional properties is of paramount importance. By making use of the recently developed SpyCatcher-SpyTag chemistry, we successfully engineered protein hydrogels on the basis of engineered tandem modular elastomeric proteins. Our resultant protein hydrogels are soft but stable, and show excellent biocompatibility. As the first step, we tested the use of these hydrogels as a drug carrier, as well as in encapsulating human lung fibroblast cells. Our results demonstrate the robustness of the SpyCatcher-SpyTag chemistry, even when the SpyTag (or SpyCatcher) is flanked by folded globular domains. These results demonstrate that SpyCatcher-SpyTag chemistry can be used to engineer protein hydrogels from tandem modular elastomeric proteins that can find applications in tissue engineering, in fundamental mechano-biological studies, and as a controlled drug release vehicle.