Dynamic Control of Hydrogel Properties Via Enzymatic Reactions
Abstract
Dynamic changes to the extracellular matrix (ECM) impact many cell fate processes. The ECM can experience changes in stiffness as well as changes in composition in response to injury, development, and diseases. To better understand the role that these dynamic processes have on the cells residing within the environment, researchers have turned towards 4-dimensional (4D) hydrogel designs. These 4D hydrogels re-capitulate not only 3-dimensional (3D) matrix architectures, but also temporal changes in the physicochemical properties. The goal of this thesis was to design a unify chemistry (i.e., Sortase A (SrtA)-mediated transpeptidation) for dynamic tuning hydrogel stiffness and the presence of bioactive ligands. The first objective was to establish a tunable and cytocompatible enzymatic scheme for softening cell-laden hydrogels. Briefly, the effects of SrtA-mediated matrix cleavage were investigated using poly(ethylene glycol) (PEG)-peptide hydrogels crosslinked by SrtA-sensitive and insensitive peptides. Initially, the effects of various parameters with respect to catalytic reactions of SrtA were characterized rheologically, including enzyme and substrate concentrations, macromer content, peptide composition, and treatment time. Gel moduli pre- and post-enzyme treatment were measured to verify SrtA-mediated hydrogel softening. The cytocompatibility of SrtA-mediated gel softening system was investigated using human mesenchymal stem cell (hMSC). Upon treatment with SrtA and an oligoglycine substrate, encapsulated hMSCs exhibited extensive spreading in comparison to those within statically stiff matrices. The second objective was to establish a reversible ligand exchange system utilizing SrtA-mediated transpeptidation. SrtA-sensitive pendant ligands were immobilized within PEG hydrogels, which were treated with SrtA and an oligoglycine substrate to afford tunable removal of the pendant ligand. Through measurement of the liberated pendant peptide concentration, it was found that higher concentrations of SrtA or extending treatment times led to higher ligand removal efficiency. Finally, the effect of peptide ligand removal on cell behaviors were evaluated using NIH 3T3 fibroblasts. Fibroblasts were culture both on and within hydrogels containing SrtA-cleavable cell adhesion peptide. After treatment, both conditions led to a decrease in fibroblast spreading in comparison to non-treated gels. Overall, the utility of SrtA as versatile agent for controlling the mechanical properties and the presence of biologically active components within a hydrogel system was demonstrated. These systems could be further explored with natural-based materials to better mimic the physiological environment experienced by cells.
Degree
M.Sc.
Advisors
Li, Purdue University.
Subject Area
Design|Physiology|Cellular biology|Immunology|Mechanics|Polymer chemistry
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