Date of Award

Summer 7-29-2014

Document Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Joel Kaar

Second Advisor

Stephanie Bryant

Third Advisor

Mark Stoykovich

Abstract

Primitive animals and earl mammalian embryos have the capacity to respond to injury by regenerating the affected tissue. Most tissues in the human body, however, follow an inferior method of wound repair that finishes with an accumulation of collagen, also known as scar tissue. Scar tissue blocks progenitor cells from infiltrating the site of injury, resulting in incomplete regeneration. Slowing the fibrotic response would shift the wound repair process towards regeneration and considerably improve human health.

The primary goal of this project is to develop novel bioactive sutures for preventing fibrosis in injured skeletal muscle. This will be achieved by immobilization of collagenase enzymes on polyethylene terephthalate (PET) sutures.

Carboxylic groups were introduced on PET sutures, which were later coupled via EDC and sulfo-NHS to primary amines on the collagenases forming an amide bond. We immobilized Clostridium histolyticum collagens as well as soluble collagens, and making multiple cleavages within the triple helical region.

While trying to control the immobilization reaction, we observed that there was non-specific binding between the enzyme and the suture. Specifically, around 70% of the activity on the sutures was due to enzyme non-covalently attached to the suture, and only 30% was due to enzyme covalently attached to the suture. The hydrophobic adsorption of the enzyme to the suture was minimized by addition of 0.05% tween 20 (v/v) to the coupling and washing solutions.

We tested the stability of the collagenases immobilized on the sutures relative to the native collagenases at 37°C, and the enzymes on the sutures lost activity faster. After two hours, there was no enzyme activity on the suture, whereas the free enzyme in solution still retained more than 50% of the activity.

In order to obtain higher collagenolytic activity on the sutures, further improvement of the immobilization method is required. This can be achieved, for example, by introduction of non-natural amino acids on the enzyme that allow for site-specific conjugation of the enzyme on the suture.

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