Tunable mechanical stability and deformation response of a resilin-based elastomer

Abstract

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues, and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this Article, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G' can be easily tuned within the range of 500 Pa to 10 kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by nonmechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicating elastic modulus values from 200 to 2500 Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled to their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.

Figure 1. Schematic cross-linked resilin-based hydrogel network and images of gels

The left panel is an uncross-linked 20wt% RLP solution; the middle panel is a 20wt% RLP hydrogel with a 1:1 cross-linking ratio (amine : HMP); the right panel is a film of a 20wt% RLP hydrogel with 1:1 cross-linking ratio 20wt%.

Figure 2. Swelling ratio and water content of RLP-based hydrogels

Equilibrium swelling ratio (q) and percentage water content (%) for 20wt% RLP-based hydrogels were tested on an average of 8-repeat measurements at 1:1, 1:2 and 1:4 (lysine : HMP) cross-linking ratios, with error reported as the standard deviation. The equilibrium swelling ratios and water content values are statistically different from one another (p < 0.05).

Figure 3. Time sweep for in situ cross-linking of RLP12

Oscillatory rheology time sweep of 25wt% RLP12 1:1 (lysine : HMP) cross-linking ratio was conducted at 25°C with frequency at 6 rad/s and 1% strain for an hour. Viscoelastic properties of RLP-based hydrogels are represented via storage modulus G′ (solid phase, solid squares) and loss modulus G″ (liquid phase, open squares).

Figure 4. Oscillatory rheology frequency sweep experiments for RLP-based hydrogels

Frequency sweeps over a range of 0.1 to 100rad/s were conducted on 20wt% RLP-based hydrogels at 1:0.5, 1:1, 1:2, 1:4 and 1:5 (lysine : HMP) cross-linking ratios at 37 °C and 1% strain.

Figure 5. Tensile testing experiments on RLP-based hydrogels

Three repeated strain-loading and unloading cycles were employed for hydrated 20wt% RLP12 films at various cross-linking ratios. A, three repeats up to 30% strain at various cross-linking ratios; B, third cycle of loading and unloading (up to 30%, 60% and 100% strain) for RLP hydrogels with a 1:1 cross-linking ratio; C, third cycle of loading and unloading (up to 30%, 60% and 100% strain) for RLP hydrogels with a 1:2 cross-linking ratio; D, third cycle of loading and unloading (up to 30%, 60% and 100% strain) for RLP hydrogels of a 1:4 cross-linking ratio.

Figure 6. Strain-to-break tensile testing experiments on RLP-based hydrogels

Strain-to-break experiments were employed for hydrated 20wt% RLP12 films at 1:1, 1:2 and 1:4 cross-linking ratios.

Figure 7. Frequency dependence of the amplification factor for RLP-based hydrogels

20wt% 1:1 (lysine : HMP) cross-linking ratio RLP hydrogel disc sample was sandwiched between the plates, fit model is shown as curve and experimental results are shown as open square symbols.

Figure 8. Rheological properties of RLP-based hydrogels at elevated frequencies

Samples at 20wt% RLP12 concentration 1:1 cross-linking ratio with various dimensions are tested over a 30-150Hz frequency range. Elastic moduli are represented in solid symbols while tan(δ) values are shown as open symbols.