Pancreatic islet encapsulation within biosynthetic materials has had limited clinical success due to loss of islet function and cell death. As an alternative encapsulation material, a silk-based scaffold was developed to reestablish the islet microenvironment lost during cell isolation. Islets were encapsulated with ECM proteins (laminin and collagen IV) and mesenchymal stromal cells (MSCs), known to have immunomodulatory properties or to enhance islet cell graft survival and function. After a 7 day in vitro encapsulation, islets remained viable and maintained insulin secretion in response to glucose stimulation. Islets encapsulated with collagen IV, or laminin had increased insulin secretion at day 2 and day 7, respectively. A 3.2-fold synergistic improvement in islet insulin secretion was observed when islets were co-encapsulated with MSCs and ECM proteins. Furthermore, encapsulated islets had increased gene expression of functional genes; insulin I, insulin II, glucagon, somatostatin, and PDX-1, and lower expression of the de-differentiation genes cytokeratin 19 and vimentin compared to non-encapsulated cells. This work demonstrates that encapsulation in silk with both MSCs and ECM proteins enhances islet function and with further development may have potential as a suitable platform for islet delivery in vivo.

Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking, evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, crosslinking occurred within two minutes, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties of these gels, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications.

Monodisperse protein polymers engineered by biosynthetic techniques are well suited to serve as a basis for creating comb-like polymer architectures for biomaterial applications. We have developed a new class of linear, cationic, random-coil protein polymers designed to act as scaffolds for multivalent display. These polymers contain evenly spaced lysine residues that allow for chemical or enzymatic conjugation of pendant functional groups. Circular dichroism (CD) spectroscopy and turbidity experiments have confirmed that these proteins have a random coil structure and are soluble up to at least 65 °C. Cell viability assays suggest these constructs are non-toxic in solution up to a concentration of 100 μM. We have successfully attached a small bioactive peptide, a peptoid-peptide hybrid, a poly(ethylene glycol) (PEG) polymer, and a fluorophore to the protein polymers by chemical or enzymatic coupling, demonstrating their suitability to serve as multivalent scaffolds in solutions or as gels.

Magnetic Resonance Imaging (MRI) is a noninvasive imaging modality with high spatial and temporal resolution. Contrast agents (CAs) are frequently used to increase the contrast between tissues of interest. To increase the effectiveness of MR agents, small molecule CAs have been attached to macromolecules. We have created a family of biodegradable, macromolecular CAs based on protein polymers, allowing control over the CA properties. The protein polymers are monodisperse, random coil, and contain evenly spaced lysines that serve as reactive sites for Gd(III) chelates. The exact sequence and length of the protein can be specified, enabling controlled variation in lysine spacing and molecular weight. Relaxivity could be modulated by changing protein polymer length and lysine spacing. Relaxivities of up to ∼14 mM -1 s -1 per Gd(III) and ∼461 mM -1 s -1 per conjugate were observed. These CAs are biodegradable by incubation with plasmin, such that they can be easily excreted after use. They do not reduce cell viability, a prerequisite for future in vivo studies. The protein polymer CAs can be customized for different clinical diagnostic applications, including biomaterial tracking, as a balanced agent with high relaxivity and appropriate molar mass.

The majority of clinically used contrast agents (CAs) for magnetic resonance imaging have low relaxivities and thus require high concentrations for signal enhancement. Research has turned to multivalent, macromolecular CAs to increase CA efficiency. However, previously developed macromolecular CAs do not provide high relaxivities, have limited biocompatibility, and/or do not have a structure that is readily modifiable to tailor to particular applications. We report a new family of multivalent, biomacromolecular, genetically engineered protein polymer-based CAs; the protein backbone contains evenly spaced lysines that are derivatized with gadolinium (Gd(III)) chelators. The protein's length and repeating amino acid sequence are genetically specified. We reproducibly obtained conjugates with an average of 8 -9 Gd(III) chelators per protein. These multivalent CAs reproducibly provide a high relaxivity of 7.3 mM -1 s -1 per Gd(III) and 62.6 mM -1 s -1 per molecule. Furthermore, they can be incorporated into biomaterial hydrogels via chemical crosslinking of remaining free lysines, and provide a dramatic contrast enhancement. Thus, these protein polymer CAs could be a useful tool for following the evolution of tissue engineering scaffolds.One significant barrier to the development of new generations of biocompatible materials, particularly tissue engineering hydrogels, is an inability to non-invasively evaluate the properties and performance of the biomaterial over time (1-6). Magnetic Resonance Imaging (MRI) is capable of whole animal or human imaging at high spatial and temporal resolution, and is an ideal modality for evaluating tissue engineering scaffolds in vivo (7-11). Exogenous contrast agents (CAs) increase the relaxation rate (1/T 1 ) of water protons and therefore improve image contrast. However, current clinically used CAs have low relaxivities (3-7 mM -1 s -1 ) (12) and thus must be used at high concentrations for useful MRI signal enhancement (12).T 1 CAs provide positive contrast by employing a paramagnetic ion (typically gadolinium, Gd (III)). The efficacy of a contrast agent is dominated by three parameters: q, the number of We have designed, synthesized and characterized a macromolecular T 1 contrast agent based on artificial proteins. The backbone of these multivalent MRI contrast agents is derived from E. coli expression of monodisperse protein polymers. Gd(III) chelators were chemically conjugated to the backbone to create contrast agents that display high relaxivity in solution and when incorporated into a hydrogel. The covalent incorporation of these protein polymer contrast agents into a gel allows the potential for imaging tissue engineering scaffolds. Here, we report the design, synthesis, and characterization of a novel family of multivalent, macromolecular CAs based on genetically engineered proteins with repetitive sequences that form the backbone for subsequent chemical modification with Gd(III) chelators. These "protein polymer" CAs have high relaxivities in aqueous solution. Mo...