Abstract of the Thesis

The Development of a Smart™ Matrix to Support Robust Fibroblast Migration

Jacob Ariel Levine

Master of Science

Biomedical Engineering

State University of New York

At Stony Brook

2002

Chronic wounds are a major health problem, affecting over 1% of the U.S. population. One important theory as to the immediate cause of nonhealing wounds is that the highly proteolytic environment of a chronic ulcer breaks down the plasma fibronectin (FN) that is normally found in the blood clot. FN is a very important molecule for wound healing. It acts as a conduit, allowing activated fibroblasts from the periwound tissue to enter the clot and commence laying matrix for granulation tissue.

FN is a large dimeric protein that is subdivided into functional domains separated by proteolytically susceptible linkers. There is data suggesting that in combination, just three of these functional domains of recombinant rat FN are necessary and sufficient to support maximal migration of fibroblasts. We have been able to explore and confirm this by adapting a two- dimensional assay in which fibroblasts are embedded in an agarose droplet and migrate out over various coated human FN fragments.

To treat chronic ulcers, we envision a Smart™ matrix, composed of a crosslinked hyaluronan (HA) scaffold decorated with the three biologically active domains. This should allow for a bioresorbable scaffold that should expedite cellular infiltration into the wound bed by protecting the FN conduit from degradation. HA is of choice, not only because it forms a hydrogel when crosslinked, but it also stimulates cell migration in synergy with FN. To this end we have developed a HA migration assay in which fibroblasts migrate from collagen-coated beads into various crosslinked HA constructs, including a matrix composed of intact FN crosslinked to the HA (HAFN). The crosslinking was confirmed successful, as no free or degraded FN was detected. However, the HAFN construct exhibited cytotoxicity and there was no perceived migration.

We are still in a very early stage of product development. Although we have not yet built a FN crosslinked to HA matrix that can support fibroblast migration, we were able to come closer to that goal through our experience with this crosslinking scheme and the HA migration assay.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture