Resilin-Based Hybrid Hydrogels for Cardiovascular Tissue Engineering

Abstract

The outstanding elastomeric properties of natural resilin, an insect protein, have motivated the engineering of resilin-like polypeptides (RLPs) as a potential material for cardiovascular tissue engineering. The RLPs, which incorporate biofunctional domains for cell-matrix interactions, are cross-linked into RLP-PEG hybrid hydrogels via a Michael-type addition of cysteine residues on the RLP with vinyl sulfones of an end-functionalized multi-arm star PEG. Oscillatory rheology indicated the useful mechanical properties of these materials. Assessments of cell viability via con-focal microscopy clearly show the successful encapsulation of human aortic adventitial fibroblasts in the three-dimensional matrices and the adoption of a spread morphology following 7 days of culture.

Keywords: biomaterials; elastomers; hydrogels; mechanical properties; tissue engineering.

Figure 1

Scheme of the hydrogel formation and details of the cross-linking chemistry are presented alongside the amino acid sequence of the RLPs. Additionally, a legend indicating the resilin-like and biological domains is presented beneath the amino acid sequence. The parentheses indicate repeated sequences; in total, there are 12 repeats of the resilin sequence and two repeats of the integrin-binding domain within the larger brackets. The brackets indicate the repeated sequences of the higher molecular weight RLPs (RLP12 × = 1, RLP24 × = 2, RLP36 × = 3, RLP48 × = 4).

Figure 2

(A) Agarose gel showing the BamHI/HindIII digestion of pET28aRLP48, pET28aRLP36, pET28aRLP24, pET28aRLP12, & pET28a (left to right). The linearized pET28a plasmid is clearly resolved between 5000 and 6000 bp while the RLP genes are represented by the bands migrating at approximately 800 bp to 3200 bp. (B) Coomassie stained SDS-PAGE gel (12%) showing the purified RLP proteins, RLP48, RLP36, RLP24, & RLP12 (left to right).

Figure 3

(A) Oscillatory rheology time sweep of a 20 wt% RLP24-PEG hydrogel cross-linked at 1:1 (gray) or 3:2 (black) molar ratios of vinyl sulfone to cysteine, at 37 °C with an angular frequency of 6 rad s⁻¹ and a strain of 1%. The inset demonstrates that the storage modulus (G′, solid squares) exceeds the loss modulus (G″, open squares) within a minute. (B) Oscillatory rheology frequency sweep experiments for RLP24-PEG hydrogels. Frequency sweeps were conducted over a range from 0.1 to 100 rad s⁻¹ for 20 wt% hydrogels cross-linked at 1:1 (gray) or 3:2 (black) vinyl sulfone to cysteine at 37 °C. The closed symbols indicate the storage modulus (G′) and the open symbols indicate the loss modulus (G″).

Figure 4

Fluorescent laser scanning confocal microscopy of human aortic adventitial fibroblasts (AoAF) encapsulated in 20 wt% RLP24-PEG hydrogel cross-linked at a 3:2 vinyl sulfone to cysteine ratio, stained using Live/Dead stains. Calcein AM (Live) stain is shown in green and ethidium homodimer (Dead) stain is shown in red. Images are z-stacks of hydrogels (200 μm thick) taken at 10× magnification using a water lens. Representative images from (A) day 0, (B) day 1, (C) day 3, and (D) day 7 are presented; the AoAFs remain viable throughout the entire experiment. Initially, the cells are rounded but as the experiment progresses they begin to adopt a spread morphology.

Figure 5

Proliferation data for encapsulated human aortic adventitial fibroblasts (AoAF) in RLP24-PEG hydrogels over 7 d of cell culture. The bars represent the average number of living nuclei counted at a given time point; the error bars represent one standard deviation from the mean and the open circles represent the counts for each hydrogel analyzed (n = 3). The number of living cells was determined by finding the difference between the number of nuclei stained by membrane-permeable Draq5 and the number of nuclei stained by membrane-impermeable ethidium homodimer. Fluorescent laser scanning confocal microscopy was used to analyze four adjacent z-stacks that were 1800 μm × 1800 μm × 400 μm (length × width × depth) for three different hydrogels at each time point. Volocity was used to count the cell nuclei for both the Draq5 and the ethidium homodimer channels. The results show that the number of nuclei remains relatively stable and that by day 7 the cells may be beginning to proliferate.