Current protocols to encapsulate cells within physical hydrogels require substantial changes in environmental conditions (pH, temperature, or ionic strength) to initiate gelation. These conditions can be detrimental to cells and are often difficult to reproduce, therefore complicating their use in clinical settings. We report the development of a two-component, molecular-recognition gelation strategy that enables cell encapsulation without environmental triggers. Instead, the two components, which contain multiple repeats of WW and proline-rich peptide domains, undergo a sol-gel phase transition upon simple mixing and hetero-assembly of the peptide domains. We term these materials mixing-induced, two-component hydrogels. Our results demonstrate use of the WW and proline-rich domains in protein-engineered materials and expand the library of peptides successfully designed into engineered proteins. Because both of these association domains are normally found intracellularly, their molecular recognition is not disrupted by the presence of additional biomolecules in the extracellular milieu, thereby enabling reproducible encapsulation of multiple cell types, including PC-12 neuronal-like cells, human umbilical vein endothelial cells, and murine adult neural stem cells. Precise variations in the molecular-level design of the two components including (i) the frequency of repeated association domains per chain and (ii) the association energy between domains enable tailoring of the hydrogel viscoelasticity to achieve plateau shear moduli ranging from Ϸ9 to 50 Pa. Because of the transient physical crosslinks that form between association domains, these hydrogels are shear-thinning, injectable, and self-healing. Neural stem cells encapsulated in the hydrogels form stable three-dimensional cultures that continue to self-renew, differentiate, and sprout extended neurites.biomaterial ͉ cell transplantation ͉ protein engineering

Improved retention of transplanted stem cells is achieved through minimally invasive delivery in MITCH, a Mixing-Induced Two-Component Hydrogel engineered to possess shear-thinning and self-healing thixotropic properties. MITCH, an ideal injectable cell-delivery vehicle, supports 3D stem cell culture, resulting in high cell viability and physiologically relevant cell morphology.

Cytokines, chemokines, and growth factors were assayed from the supernatants of monocytes and macrophages cultured on common biomaterials with a range of surface chemistries. TNF-α, MCP-1, MIP-1α, IL-8, IL-6, IL-1β, VEGF, IL-1ra, and IL-10 were measured from monocyte/macrophage cultures at different stages of activation and differentiation seeded onto polyethylene, poly-urethane, expanded polytetrafluoroethylene, polymethyl methacrylate, and a hydrogel copolymer of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol acrylate in tissue culture polystyrene (TCPS) plates. Empty TCPS wells and organo-tin polyvinyl chloride served as “blanks” and positive controls, respectively. Results showed an overall increase in cytokine, chemokine, and growth factor production as monocytes are activated or differentiated into macrophages and that proinflammatory and anti-wound healing cytokines and chemokines dominate this profile. However, cytokine production was only modestly affected by the surface chemistry of these four stable and noncytotoxic biomaterials.

Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

Recently, supramolecular hydrogels assembled through nonspecific interactions between polymers and nanoparticles (termed PNP systems) were reported to have rapid shear-thinning and self-healing properties amenable for cell-delivery applications in regenerative medicine. Here, we introduce protein engineering concepts into the design of a new family of PNP hydrogels to enable direct control over the polymer−nanoparticle interactions using peptide-based molecular recognition motifs. Specifically, we have designed a bifunctional peptide that induces supramolecular hydrogel assembly between hydroxy apatite nanoparticles and an engineered, recombinant protein. We demonstrate that this supramolecular assembly critically requires molecular recognition, as no assembly is observed in the presence of control peptides with a scrambled amino acid sequence. Titration of the bifunctional peptide enables direct control over the number of physical cross-links within the system and hence the resulting hydrogel mechanical properties. As with previous PNP systems, these materials are rapidly shear-thinning and self-healing. As proof-of-concept, we demonstrate that these materials are suitable for therapeutic cell delivery applications in a preclinical murine calvarial defect model.