Mechanical characterization of ECM-mimetic hydrogel based on heparin -peptide interactions

Kyung Jae Jeong, Purdue University

Abstract

The motivation behind this body of work was the development of biohybrid materials that better mimic the mechanical properties of the extracellular matrix (ECM). Cell-ECM interactions play crucial roles in controlling cell migration, proliferation and differentiation. Lately, the mechanical properties of ECM, independent of its chemical nature, have been recognized as important factors in controlling such cellular functions through physical couplings that link the ECM to the cell cytoskeleton, thus allowing for direct force transduction. The mechanical properties of ECM may be modulated by the combination of covalent and physical interactions between the ECM constituent molecules. Previously, in our laboratory, an ECM-mimetic hydrogel made of multi-arm poly (ethylene glycol) (PEG) was developed. In this hydrogel, vinyl sulfone-derivatized PEG molecules (PEG-VS) were crosslinked by a dithiol-containing peptide, which served as a covalent crosslinker, and by the binding between heparin and heparin binding peptide (HBP), which served as a physical crosslinker. This hydrogel exhibited the rheological properties of both covalently and physically crosslinked hydrogels. In this work, we examined whether covalent and physical interactions would independently contribute to the bulk mechanical properties when they coexist within a gel. Prior to carrying out the mechanical measurements, a novel assay to probe the affinity between heparin and HBP using gold nanoparticles was developed to facilitate the search for proper peptide sequences. This assay utilizes the rapid aggregation of gold nanoparticles in the presence of HBP and its retardation in the presence of a large excess of heparin. Using this label-free and easy-to-use method, dissociation constants (Kd) were measured for various HBPs and the results were validated by comparing the results with those obtained by affinity capillary electrophoresis (ACE). Using the peptide, selected by the gold nanoparticle assay, the dynamic mechanical properties of PEG hydrogels of varying compositions of covalent and physical crosslinkers were characterized by rheometry. Strikingly, the presence of covalent crosslinks enhanced the contribution from the physical crosslinks to the storage modulus by three orders of magnitude. Two potential mechanisms are proposed to explain this unusual phenomenon. In the absence of covalent crosslinks, the average size of PEG clusters is small due to the weak physical interactions between heparin and HBP. Under this condition, each cluster relaxes the stress too quickly that the physical interactions cannot contribute to the storage modulus. Upon the addition of covalent crosslinkers, the average cluster size increases dramatically, resulting in a much extended stress relaxation time, under which condition physical interactions can participate in bearing the mechanical load as long as the lifetime of the binding between heparin and HBP is longer than the time scale of the experiment. Stress relaxation time measurements by rheometer and the measurement of the lifetime of the binding by AFM force spectroscopy confirmed this hypothesis. Another possible explanation is based on the ‘macromolecular confinement’ effect. It is known that the biological binding and the stability of protein folding are greatly enhanced in confined spaces. Since the binding between heparin and HBP takes place within confined spaces defined by the covalent mesh, the macromolecular confinement effect is expected to affect the equilibrium of the binding Kd measurement using quartz crystal microbalance (QCM) confirmed that the binding inside the hydrogel was enhanced by an order of magnitude. Since ECM is also made of the combination of covalent and physical interaction between various structural proteins and polysaccharides, it is expected that the findings of this work have many important implications in cell biology and tissue engineering.

Degree

Ph.D.

Advisors

Panitch, Purdue University.

Subject Area

Biomedical engineering|Chemical engineering

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