Soft Hydrogel Inspired by Elastomeric Proteins

Abstract

Elastin polypeptides based on -VPGVG- repeated motifs are widely used in the production of biomaterials because they are stimuli-responsive systems. On the other hand, glycine-rich sequences, mainly present in tropoelastin terminal domains, are responsible for the elastin self-assembly. In a previous study, we have recombinantly expressed a chimeric polypeptide, named resilin, elastin, and collagen (REC), inspired by glycine-rich motifs of elastin and containing resilin and collagen sequences as well. Herein, a three-block polypeptide, named (REC)₃, was expressed starting from the previous monomer gene by introducing key modifications in the sequence. The choice was mandatory because the uneven distribution of the cross-linking sites in the monomer precluded the hydrogel production. In this work, the cross-linked polypeptide appeared as a soft hydrogel, as assessed by rheology, and the linear un-cross-linked trimer self-aggregated more rapidly than the REC monomer. The absence of cell-adhesive sequences did not affect cell viability, while it was functional to the production of a material presenting antiadhesive properties useful in the integration of synthetic devices in the body and preventing the invasion of cells.

Keywords: antiadhesive materials; circular dichroism; cytocompatibility; elastin; hydrogel.

Figure 1

Schematic representation of the primary structure of the (REC)₃ polypeptide.

Figure 2

12% SDS-PAGE analysis of (REC)₃ production screening (A) and purification process (B). (A) Total protein fraction analysis of E. coli BLR(DE3) after overnight induction in a modified TB medium. Lane M: protein marker; lane Ø—negative control, untransformed BLR(DE3); and Lanes 1–5, five transformed colonies were randomly selected and analyzed. The red marker highlights the (REC)₃ polypeptide production by different transformants. (B) Increasing amounts of the purified (REC)₃ polypeptide were evaluated: 5 μg (lane 1), 10 μg (lane 2), and 15 μg (lane 3); M: protein marker, the molecular weight of bands is indicated.

Figure 3

(a) CD spectra of the (REC)₃ polypeptide in PBS (filled symbols) and TFE (open symbols) at the indicated temperatures: 0 °C (squares); 25 °C (circles); 37 °C (diamonds); and 60 °C (triangles); (b) CD spectra of the REC polypeptide in an aqueous solution (filled symbols) and TFE (open symbols) at the indicated temperatures: 0 °C (squares); 25 °C (circles); and 60 °C (triangles).

Figure 4

TEM micrographs of (REC)₃ incubated at 37 °C after drop deposition (a) and after 24 h (b).

Figure 5

AFM images of (REC)₃ incubated at 37 °C after 0 (a) and 24 (b) h.

Figure 6

(a) Hydrogel; (b) SEM image of the hydrogel, where bar represents 30 μm; (c) evolution of G′ and G″ with frequency at 1% strain: average storage modulus (black) and loss modulus (red); and (d) dependence of the complex modulus magnitude with f^1/2.

Figure 7

Viability percentage as the metabolic activity of HUVECs and MSCs with respect to untreated cells in the same conditions. Cells were incubated with 5 mg/mL (REC)₃ for 24 or 72 h. Metabolic active cell numbers were measured, each sample four-fold, using the Alamar Blue assay kit in three independent experiments. No statistically significant differences are observed. The error bars represent the standard deviation.

Figure 8

Viability of HUVECs and MSCs cultured on surfaces coated with (REC)₃ solutions at 5 or 10 mg/mL concentrations. Histogram represents the percentages of live cells after 4 days of incubation with respect to live cells cultured on a standard tissue culture support. The error bars correspond to the standard deviation.

Figure 9

Panels A (10×) and B (20×): representative images of HUVECs cultured on fibronectin (positive control), 5 mg/mL or 10 mg/mL (REC)₃-coated surfaces. Panels C (10×) and D (20×): representative images of hMSCs cultured on fibronectin (positive control), 5 mg/mL or 10 mg/mL (REC)₃-coated surfaces. The living cells fluoresce green, whereas dead cells with compromised membranes fluoresce red.