Resilin-like polypetide-based microstructured hydrogels via aqueous-based liquid-liquid phase separation for tissue engineering applications
Date
2018
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Hydrogels provide mechanical support and a hydrated environment that offer good cytocompatibility and controlled release of molecules, and myriad hydrogels thus have been studied for biomedical applications. Recent research has increasinglyfocused on multicomponent hydrogels that better capture the multifunctional andmicrostructural nature of native biological environments. ☐ Multiple approaches to generate microstructured hydrogels have emerged in order to control microscale properties for applications ranging from mechanical reinforcement to regenerative medicine. In this thesis, we introduce new heterogeneous hybrid hydrogels comprising emerging resilin-like polypeptides (RLPs) and poly(ethylene glycol) (PEG). Phase diagrams of the RLP/PEG system were generated in order to define solution parameters that would yield micron-scale domains in the hydrogels. The hydrogels can be engineered with controlled microstructure and distinct micromechanical properties via the liquid-liquid phase separation (LLPS) of aqueous solutions of the RLPs and PEG. The microstructure in the hydrogels was captured by crosslinking a phase-separated RLP and PEG solution via a Mannich-type reaction with the crosslinker tris(hydroxymethyl phosphine) (THP). The production of RLP-rich domains and PEG-rich matrix was confirmed via confocal microscopy. The hydrogel mechanical properties were assessed via oscillatory rheology and atomic force microscopy (AFM), with the hydrogels exhibiting a moderate bulk shear storage modulus (ca. 600 Pa) and micromechanical properties of the domains (Young’s modulus ca. 13 kPa) that were distinct from those of the matrix (ca. 6 kPa). These results demonstrate that tuning the parameters of the aqueous-aqueous phase-separated RLP/PEG solutions provides a simple, straightforward methodology for fabricating microstructured protein-containing hydrogels, without extensive material processing or purification. ☐ Despite the range of such microstructured materials described, few methods permit independent control over microstructure and microscale mechanics by precisely controlled, single-step processing methods. We further reported a photo-triggered crosslinking methodology that traps microstructures in LLPS solutions of RLP and PEG. RLP-rich domains of various diameters could be trapped in a PEG continuous phase, with the kinetics of domain maturation dependent on the degree of acrylation. The chemical composition of both hydrogel phases over time was assessed via in situ hyperspectral coherent Raman microscopy, with equilibrium concentrations consistent with the compositions derived from NMR-measured coexistence curves. Atomic force microscopy revealed that the local mechanical properties of the two phases evolved over time, even as the bulk modulus of the material was constant, showing that our strategy permits control of mechanical properties on micrometer length scales, of relevance in generating mechanically robust materials for a range of applications. The successful encapsulation, localization, and survival of stem cells (hMSCs) was demonstrated and suggests the potential application of phase-separated RLP/PEG hydrogels in regenerative medicine applications. ☐ Furthermore, micromechanical properties of RLP-PEG microstructuredhydrogels were characterized via oscillatory shear rheology, small-strain microindentation, and large-strain indentation and fracture. Oscillatory shear rheology and small-strain microindentation measured the small-strain elastic response of RLP-PEG hydrogels. The elastic moduli calculated from rheology were comparable with the elastic moduli obtained from microindentation. Repeated cyclic loading and unloading microindentation revealed high resilience values (>85%) for RLP-PEG hydrogels even up to 80% strain. Large-strain puncture under a confocal microscope enabled the visualization of the microstructured hydrogel under indentation and deformation of RLP-rich domains. Puncture experiments also characterized the mechanical response and effective elastic moduli of the RLP-PEG, RLP-rich and PEG-rich hydrogels. The impact of spherical indenter sizes on puncture mechanics were also evaluated and extracted a fracture energy and maximum stress of the microstructured RLP-PEG hydrogels. Microstructured RLP-PEG maintain excellent mechanical properties and biocompatibility suggesting their potential in tissueengineering applications.