Research Article: Microstructured Elastomer‐PEG Hydrogels via Kinetic Capture of Aqueous Liquid–Liquid Phase Separation

Date Published: March 12, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Hang Kuen Lau, Alexandra Paul, Ishnoor Sidhu, Linqing Li, Chandran R. Sabanayagam, Sapun H. Parekh, Kristi L. Kiick.


Heterogeneous hydrogels with desired matrix complexity are studied for a variety of biomimetic materials. 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. Here, a phototriggered crosslinking methodology that traps microstructures in liquid–liquid phase‐separated solutions of a highly elastomeric resilin‐like polypeptide (RLP) and poly(ethylene glycol) (PEG) is reported. RLP‐rich domains of various diameters can 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 is assessed via in situ hyperspectral coherent Raman microscopy, with equilibrium concentrations consistent with the compositions derived from NMR‐measured coexistence curves. Atomic force microscopy reveals that the local mechanical properties of the two phases evolve over time, even as the bulk modulus of the material remains constant, showing that the strategy permits control of mechanical properties on micrometer length scales, of relevance in generating mechanically robust materials for a range of applications. As one example, the successful encapsulation, localization, and survival of primary cells are demonstrated and suggest the potential application of phase‐separated RLP‐PEG hydrogels in regenerative medicine applications.

Partial Text

The heterogeneity and biophysical properties of native extracellular matrix (ECM) are essential in regulating cell behavior and guiding tissue regeneration. Microstructured hydrogels that mimic the complexity of ECM thus have emerged as useful materials for controlling material mechanical properties, diffusion of macro‐ and biomolecules, and mammalian cell behavior.1, 2, 3, 4, 5 A variety of evidence suggests that heterogeneity in hydrogels can promote cell growth and organization in 3D; indeed, macroporous scaffolds and hydrogels containing degradable microparticles have been shown to promote osteogenic differentiation in vitro and bone tissue formation in vivo.6, 7, 8 Furthermore, the mechanical properties of hydrogel materials, including stiffness, elasticity, and viscoelastic properties, impact the differentiation of mesenchymal stem cells as well as tissue regeneration.9, 10, 11, 12, 13 Hydrogel geometries and surface curvature also influence cell morphology and migration, thus providing additional materials handles for regulating gene expression and cell functions,14, 15 independently of bulk mechanical properties. These studies together indicate the importance of engineering hydrogel microstructures with independently tunable microscale structure and micromechanical properties for controlling cell behavior. Accordingly, multiple production strategies have been pursued, such as photopatterning,16, 17, 18, 19, 20 selective degradation,16, 21, 22 and the incorporation of microparticles3, 23 synthesized via emulsion polymerization24, 25, 26, 27 and/or microfluidic28 technologies. However, these methods suffer from either low throughput, as in the case of photopatterning, or multiple processing steps, as in particle fabrication.

Photo‐crosslinking methods were exploited as a facile method to capture morphologically, chemically, and mechanically distinct phases in microstructured hydrogels during LLPS of RLP‐Ac/PEG‐4Ac solutions. Evaluation of the LLPS for RLPs with various degrees of acrylamide functionalization established that equilibrium phase diagrams were not significantly affected by the degree of functionalization. Photo‐triggered crosslinking of RLP‐Ac/PEG‐4Ac during the phase separation permitted the production of RLP‐PEG hydrogels with RLP‐rich domains with various diameters; a higher degree of RLP acrylation reduced the rate of domain growth, presumably by increased miscibility mediated by hydrophobic interactions between the RLP‐6Ac and PEG‐4Ac. Controllable photo‐crosslinking permits the modulation of the modulus via UV exposure time, allowing for on‐demand and independent tuning of microstructure and mechanical properties. Significant differences in the compositions (and thus mechanical properties) of the developing domains and continuous phase were indicated to occur only after 10 min of phase separation, as indicated by BCARS microscopy and AFM; interestingly, the microstructured matrices exhibit bulk mechanical properties that correspond to the rule of mixtures theory and do not vary over time. Furthermore, the materials demonstrated spatial localization of multiple cell types, at high viabilities, around RLP‐rich domains. Overall, the LLPS of RLP‐Ac/PEG‐4Ac solutions, when captured via photo‐crosslinking, permits independent tuning of the microstructure and micromechanical properties of hydrogels that can be used to design complex materials for biomedical and other applications. The high cell viability and capability to guide cell organization within the microstructured hydrogels indicates their potential use in regenerative medicine applications.

Materials: Chemically competent cells of Escherichia coli strain M15[pREP4] (for transformation of recombinant plasmids) and Ni‐NTA agarose resin (for protein purification) were purchased from Qiagen (Valencia, CA). Acrylate‐terminated 4‐arm (20 kDa) PEG was purchased from Jemken. PBS was purchased from Mediatect (Manassas, VA). Deuterium oxide and NMR solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). All other chemicals were obtained from Sigma‐Aldrich (St. Louis, MO) or Fisher Scientific (Waltham, MA) and were used as received unless otherwise noted.

The authors declare no conflict of interest.




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