Recombinant Resilin-Based Bioelastomers for Regenerative Medicine Applications

Abstract

The outstanding elasticity, excellent resilience at high-frequency, and hydrophilic capacity of natural resilin have motivated investigations of recombinant resilin-based biomaterials as a new class of bio-elastomers in the engineering of mechanically active tissues. Accordingly, here the comprehensive characterization of modular resilin-like polypeptide (RLP) hydrogels is presented and their suitability as a novel biomaterial for in vivo applications is introduced. Oscillatory rheology confirmed that a full suite of the RLPs can be rapidly cross-linked upon addition of the tris(hydroxymethyl phosphine) cross-linker, achieving similar in situ shear storage moduli (20 k ± 3.5 Pa) across various material compositions. Uniaxial stress relaxation tensile testing of hydrated RLP hydrogels under cyclic loading and unloading showed negligible stress reduction and hysteresis, superior reversible extensibility, and high resilience with Young's moduli of 30 ± 7.4 kPa. RLP hydrogels containing MMP-sensitive domains are susceptible to enzymatic degradation by matrix metalloproteinase-1 (MMP-1). Cell culture studies revealed that RLP-based hydrogels supported the attachment and spreading (2D) of human mesenchymal stem cells and did not activate cultured macrophages. Subcutaneous transplantation of RLP hydrogels in a rat model, which to our knowledge is the first such reported in vivo analysis of RLP-based hydrogels, illustrated that these materials do not elicit a significant inflammatory response, suggesting their potential as materials for tissue engineering applications with targets of mechanically demanding tissues such as vocal fold and cardiovascular tissues.

Keywords: bio-elastomer; hydrogel; resilin-like polypeptide; tissue engineering; vocal fold.

Figure 1. Representative uniaxial stress relaxation behavior of compositionally different RLP hydrogels at 60% strain

All hydrogels were formed at a polypeptide concentration of 20wt%, with a 1:1 (amine:hydroxyl) cross-linking ratio. Three repeats of each hydrogel composition were tested and the results are reproducible with less than 10% difference among multiple sample repeats observed.

Figure 2. Uniaxial tensile testing of RLP-based hydrogels

(a) Three repeats of uniaxial cyclic tensile testing up to 30% strain at various RLP hydrogel compositions, (b) third cycle of loading and unloading tensile testing up to 100% strain for 3 different RLP hydrogel compositions, and (c) resilience values of the third cycle of loading and unloading at 30%, 60% and 100% strains for various RLP hydrogel compositions.

Figure 2. Uniaxial tensile testing of RLP-based hydrogels

(a) Three repeats of uniaxial cyclic tensile testing up to 30% strain at various RLP hydrogel compositions, (b) third cycle of loading and unloading tensile testing up to 100% strain for 3 different RLP hydrogel compositions, and (c) resilience values of the third cycle of loading and unloading at 30%, 60% and 100% strains for various RLP hydrogel compositions.

Figure 2. Uniaxial tensile testing of RLP-based hydrogels

(a) Three repeats of uniaxial cyclic tensile testing up to 30% strain at various RLP hydrogel compositions, (b) third cycle of loading and unloading tensile testing up to 100% strain for 3 different RLP hydrogel compositions, and (c) resilience values of the third cycle of loading and unloading at 30%, 60% and 100% strains for various RLP hydrogel compositions.

Figure 3. Enzymatic degradation of RLP and RLP-MMP hydrogels monitored via oscillatory rheology

Time sweep experiments for RLP-MMP (circle) and RLP (square) hydrogels incubated in the presence of 200nM MMP-1, and an RLP-MMP hydrogel incubated without MMP-1 (diamond). All hydrogels were formed at a protein concentration of 20wt%, with a 1:1 (amine : hydroxyl) cross-linking ratio, and incubated at 37°C in 2mL PBS buffer (pH 7.4). A bath volume of 2 mL (50-fold greater than the volume of the hydrogel) provided an adequate sink so that the pH and MMP-1 enzyme concentration were unaffected during the experiment.

Figure 4. Immunochemical analysis of hMSCs stained, after 72 hours, for nuclei, vinculin, and actin cytoskeleton, on the surface of 20wt% RLP hydrogels

(a) 100% RLP, (b) 100% RLP-RDG, (c) 50% RLP and 50% RLP-RGD and (d) 100% RLP-RGD. All hydrogels were crosslinked at a 1:1 ratio of amine : hydroxyl groups. Data for each hydrogel composition is separated into 4 panels: (1) Cell nuclei counterstained by Draq5 (blue); (2) Focal adhesion sites visualized (green) by treatment with anti-vinculin and a FITC-labeled secondary antibody; (3) F-actin filaments visualized (red) by treatment with TRITC-phalloidin; and (4) the merged image of the triply stained hydrogels (Draq5, vinculin and TRITC-phalloidin).

Figure 4. Immunochemical analysis of hMSCs stained, after 72 hours, for nuclei, vinculin, and actin cytoskeleton, on the surface of 20wt% RLP hydrogels

(a) 100% RLP, (b) 100% RLP-RDG, (c) 50% RLP and 50% RLP-RGD and (d) 100% RLP-RGD. All hydrogels were crosslinked at a 1:1 ratio of amine : hydroxyl groups. Data for each hydrogel composition is separated into 4 panels: (1) Cell nuclei counterstained by Draq5 (blue); (2) Focal adhesion sites visualized (green) by treatment with anti-vinculin and a FITC-labeled secondary antibody; (3) F-actin filaments visualized (red) by treatment with TRITC-phalloidin; and (4) the merged image of the triply stained hydrogels (Draq5, vinculin and TRITC-phalloidin).

Figure 4. Immunochemical analysis of hMSCs stained, after 72 hours, for nuclei, vinculin, and actin cytoskeleton, on the surface of 20wt% RLP hydrogels

(a) 100% RLP, (b) 100% RLP-RDG, (c) 50% RLP and 50% RLP-RGD and (d) 100% RLP-RGD. All hydrogels were crosslinked at a 1:1 ratio of amine : hydroxyl groups. Data for each hydrogel composition is separated into 4 panels: (1) Cell nuclei counterstained by Draq5 (blue); (2) Focal adhesion sites visualized (green) by treatment with anti-vinculin and a FITC-labeled secondary antibody; (3) F-actin filaments visualized (red) by treatment with TRITC-phalloidin; and (4) the merged image of the triply stained hydrogels (Draq5, vinculin and TRITC-phalloidin).

Figure 4. Immunochemical analysis of hMSCs stained, after 72 hours, for nuclei, vinculin, and actin cytoskeleton, on the surface of 20wt% RLP hydrogels

(a) 100% RLP, (b) 100% RLP-RDG, (c) 50% RLP and 50% RLP-RGD and (d) 100% RLP-RGD. All hydrogels were crosslinked at a 1:1 ratio of amine : hydroxyl groups. Data for each hydrogel composition is separated into 4 panels: (1) Cell nuclei counterstained by Draq5 (blue); (2) Focal adhesion sites visualized (green) by treatment with anti-vinculin and a FITC-labeled secondary antibody; (3) F-actin filaments visualized (red) by treatment with TRITC-phalloidin; and (4) the merged image of the triply stained hydrogels (Draq5, vinculin and TRITC-phalloidin).

Figure 5. Activation of RAW 264.7 macrophages incubated for 8 hours on a 20wt% THP-crosslinked RLP only hydrogel, measured through production of TNF-α

The same number of RAW264.7 murine macrophages were cultured on the surface of TCPS supplemented with 1ng/mL LPS derived from E coli. as a positive control, on the surface of TCPS without LPS as negative control, and directly on the surface of THP cross-linked RLP hydrogels (20wt%). Macrophages cultured on these surfaces and all hydrogels showed 100% viability (not shown). Error bars correspond to standard deviation from the mean; the TNF-α expression is not statistically different between TCPS and RLP hydrogels; *(p<0.0001) the difference is statistically significant between the indicated groups determined via Student’s t-test analysis.

Figure 6. Subcutaneous transplantation of 20wt% RLP hydrogels in Sprague Dawley (SD) rats sacrificed after one week

Tissue samples were stained and histological images are presented with hematoxylin and eosin (H&E), cluster of differentiation 3 (CD3) and cluster of differentiation 31 (CD31) staining. The yellow box highlights the location of gel transplantation and the remnants of RLP-hydrogel after one week post transplantation.

Scheme 1

Schematic of RLP-based hydrogels with various material compositions.