Elastin‐like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function

Abstract

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell‐applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin‐like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine‐modified ELP (ELP‐HYD) and aldehyde/benzaldehyde‐modified polyethylene glycol (PEG‐ALD/PEG‐BZA). The reversible DCC crosslinks in ELP‐PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast‐relaxing or slow‐relaxing hydrogels with a range of stiffness (500–3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two‐dimensional substrates, on which ECs exhibited greater cell spreading on fast‐relaxing hydrogels up through 3 days, compared with slow‐relaxing hydrogels at the same stiffness. In three‐dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast‐relaxing, low‐stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast‐relaxing, low‐stiffness hydrogel produced significantly more vascularization compared with the slow‐relaxing, low‐stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast‐relaxing, low‐stiffness hydrogels supported the highest capillary density in vivo.

A Library of Elastin-like Proteins with Tunable Matrix Ligands for In Vitro 3D Neural Cell Culture

“…Our analysis confirms that all seven HELP formulations containing the seven different cell-instructive ligands have statistically similar shear moduli (Figure A,B). The plateau shear moduli averages ∼200 Pa, which is within the range reported to be supportive of neurite outgrowth. ,, An additional aspect of this cross-linking strategy is that hydrazone bonds result in stress-relaxing hydrogels due to their dynamic exchange kinetics. , Here, the matrix stress relaxation rate, t 0.5 , is quantified as the time required to reach half of the initial stress under a constant 10% strain. Similar to our previous work with HELP gels, all seven of the HELP formulations display slow stress relaxation behavior, with t 0.5 > 12 h (Figure C).…”

“…7,69,70 An additional aspect of this cross-linking strategy is that hydrazone bonds result in stress-relaxing hydrogels due to their dynamic exchange kinetics. 71,72 Here, the matrix stress relaxation rate, t 0.5 , is quantified as the time required to reach half of the initial stress under a constant 10% strain. Similar to our previous work with HELP gels, 31 all seven of the HELP formulations display slow stress relaxation behavior, with t 0.5 > 12 h (Figure 5C).…”

Transient Competitors to Modulate Dynamic Covalent Cross-Linking of Recombinant Hydrogels

“…Here, HA and ELP are modified along their backbone with benzaldehyde (HA-BZA; confirmed by NMR, Figure S2) or hydrazine (ELP-HYD; confirmed by NMR, Figure S3), respectively (Figure C). Combining the chemically modified HA and ELP results in a hydrazone cross-linked HELP hydrogel that is both biologically active and stable for extended culture. ,,, Using this system, we demonstrated the use of hydrazinoacetic acid as a competitor to transiently disrupt cross-linking density for the bioprinting of HELP …”

“…Combining the chemically modified HA and ELP results in a hydrazone cross-linked HELP hydrogel that is both biologically active and stable for extended culture. 41,49,50,56 Using this system, we demonstrated the use of hydrazinoacetic acid as a competitor to transiently disrupt cross-linking density for the bioprinting of HELP. 51 Inspired by the use of an alkyl hydrazine competitor to alter hydrogel network properties, we recognized the potential to utilize the side group of the competitor to modulate the reaction kinetics and fine-tune the equilibrium cross-linking density.…”

Cell Microencapsulation Within Engineered Hyaluronan Elastin‐Like Protein (HELP) Hydrogels

“…Cells can be characterized at different time points as desired; for example, the 3D culture is compatible with common assays including characterization of cell viability (live/dead staining), DNA quantification, metabolism, gene/protein expression, and morphology (Hunt et al, 2021;LeSavage et al, 2022;Navarro et al, 2022;Shayan et al, 2023;Wang et al, 2017).…”