Transglutaminase enzymes are ubiquitous Ca2+-dependent enzymes that catalyze the formation of crosslinks between glutamine and lysine residues of proteins. Extensive transglutaminase-mediated crosslinking of soluble proteins is believed to be responsible for rapid physical gelation of certain biological fluids. A common biological strategy for regulating the activity of transglutaminase enzymes is control of intracellular and extracellular Ca 2+ concentration, mediated by lipid bilayer membranes. Stimuli-responsive synthetic lipid vesicles offer a unique opportunity to regulate transglutaminase-mediated gelation by sequestering and then releasing enzyme-activating ions such as Ca 2+. We hypothesize that Ca 2+ release from temperature or light sensitive liposomes can be used to trigger TG-mediated crosslinking of peptide-modified polymers to form hydrogels suitable for use as tissue adhesives and for injectable tissue engineering. In this study, combinatorial chemistry will be employed to synthesize large peptide libraries from which short peptide substrates of transglutaminase enzymes will be identified. The peptide substrates will be covalently linked to biocompatible polymers, and the TG-catalyzed crosslinking of the polymers into hydrogels will be studied in an effort to formulate injectable solutions that undergo rapid gelation in situ. Stimuli-responsive liposomes will be utilized to trigger calcium activation of enzyme-catalyzed gelation with the goal of developing thermal and light triggered gelation for clinical use. The tissue adhesive potential of these hydrogels will be assessed by measuring the force required to separate articular cartilage surfaces bonded together by in-situ formed hydrogels, and in vitro and in vivo studies will be performed to evaluate the potential of chondrocyte-containing injectable polymer hydrogels to support the formation of cartilage tissue.
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