Resilin-like polypeptide-based elastomeric biomaterials for vocal fold tissue engineering

Elastomeric proteins are characterized by their large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Motivated by their unique mechanical properties, there has been tremendous research in understanding and manipulating elastomeric polypeptides, with most work conducted on the natural elastins but more recent work on an expanded set of polypeptide elastomers. Facilitated by biosynthetic strategies, it has been possible to manipulate the physical properties, conformational, and mechanical properties of these materials. Detailed understanding of the roles and organization of the natural structural proteins has permitted the design of elastomeric materials with engineered properties, and has thus expanded the scope of applications from elucidation of the mechanisms of elasticity to the development of high performance biomaterials and tissue engineering substrates. Natural resilin, a new candidate of rubber-like protein found in the specialized compartments of most arthropods, possesses superior mechanical properties such as low stiffness, high resilience and effective energy storage. Recombinantly engineered resilin-like polypeptides (RLPs) that possess the favorable attributes of native resilin would be attractive candidates for the modular design of biomaterials to engineer mechanically active vocal fold tissues. Towards this goal, a novel, modular designed RLP bearing 12 repeats of the putative resilin consensus motif from the first exon of the Drosophila CG15920 gene was strategically combined with biological domains for cell adhesion, proteolytic degradation, and heparin immobilization. The incorporation lysine residues in this RLP enables fast gel formation through Mannich-type reaction with the hydroxyl functional groups from the zero-length cross-linker (beta-[tris(hydroxymethyl)phosphine]propionic acid) (THPP). Detailed mechanical properties of this RLP-based hydrogels were characterized via dynamic oscillatory rheology, uniaxial tensile testing and high-frequency torsional wave analysis with results revealing the strong dependence of the mechanical properties on the extent of cross-linking but with storage moduli relevant to vocal fold tissues as well as significantly increased resilience similar to natural resilin and other synthetic RLPs. Moreover, RLP-based hydrogel facilitated the survival and proliferation of NIH 3T3 fibroblasts in vitro, further demonstrating its potential as a scaffold for tissue engineering applications. The ability to independently tailor specific cell-matrix interaction through changing multiple properties of a scaffold holds critical aspects in regulating cellular responses and guiding cell fate. In response to the original design of RLP12, which lacks the variation of relative concentration of each biological domain, we expanded the versatility of previous RLP12 to multiple RLP-based constructs, in which each construct contains the essentially the same 12 repetitive resilin consensus motifs but bears a different biologically active module. Via this simple and straightforward approach, it is possible to independently modulate the concentrations of cell-binding, MMP-sensitive, and polysaccharide-sequestration domains in hydrogels of selected mechanical properties; thus the biological composition can be decoupled from the mechanical properties in the materials comprising mixtures of the various RLPs. The high purity, molecular weight and correct compositions of each new polypeptide have been confirmed via high performance liquid chromatography (HPLC), sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), and amino acid analysis. These RLP-based polypeptides exhibit largely random-coil conformation, both in solution and in the cross-linked hydrogels, as indicated by circular dichroic and infrared spectroscopic analyses. Upon exposure to matrix metalloproteinase (MMP-1), RLP constructs containing MMP-sensitive domains exhibited enzymatic degradation within 48 hours while those without the MMP-sensitive substrates remained intact during the entire incubation period. Biocompatible Mannich-type condensation reaction between thiamine pyrophosphate and lysines is employed in additional interactions to create elastomeric RLP-based biomaterials. Hydrogels with various compositions, within a range of elastic moduli (1-25 kPa), yield comparable mechanical features (e.g. storage moduli, stress relaxation, creep, stain-to-break and resilient properties), however, they exhibit completely different biological responses resulted from the identity and concentration of the biological active domains presented in the hydrogels. RLP hydrogels were able to maintain their mechanical integrity as well as the viability of encapsulated primary human mesenchymal stem cells (MSCs) up to 21 days. Preliminary assessment of the inflammatory properties of RLP hydrogels was investigated by culturing murine macrophage on the surface of the RLP-based hydrogels and the negligible secretion of a pro-inflammatory cytokine TNF-&agr; demonstrated that the RLP-hydrogels do not activate macrophages. Together, this straightforward mixing and matching strategy offers substantial opportunities for fabricating elastomeric RLP-based hydrogels as regenerative scaffold that can be systematically tailored for targeted implantation or injection in vocal fold tissue therapies.