The long- term goal of the project is to be able to treat non-healing dermal wounds through the application of a smart matrix construct to the wound bed. The current hypothesis is that the formation of a Smart™ matrix composed of crosslinked hyaluronan and three functional domains of fibronectin (cell binding domain, heparin II binding domain, and IIICS variable region) will support robust fibroblast migration in vitro and will address issues of the “ideal” Smart™ matrix for soft tissue wounds. These issues include the biocompatibility of the construct, the controlled bioresorption of the scaffold, the enhancement of wound healing as compared to the standard of care, the prevention of scar formation, the ease of application, and the manufacture and distribution of the final product at low cost to the manufacturer as well as to the consumer.

The first issue, the biocompatibility of the construct, is addressed by the constituents of the Smart™ matrix. The matrix is composed entirely of elements native to the skin during wound repair. However, biocompatibility will need to be assessed for the crosslinked state of hyaluronan, as well as for the crosslinked fibronectin fragments. The degradation byproducts of the matrix should approximate the degradation byproducts of uncrosslinked hyaluronan that is normally present in the wound, so no adverse compatibility issues are expected.

The second issue, the controlled bioresorption of the scaffold, is addressed by the extent and nature of the crosslinking involved. When the Smart™ matrix is applied to a wound, the scaffold will begin to be degraded by free hyaluronidase in the wound fluid, as well as by cells as they migrate into the scaffold. The covalent crosslinking of the hyaluronan to itself should prevent this degradation from advancing too rapidly and should allow a controlled degradation profile that can be optimized by altering the extent of crosslinking.

The third issue, the enhancement of wound healing as compared to the standard of care, is addressed by the direct comparison of the efficacy of the Smart™ matrix to the efficacy of various FDA approved methods of treatment in promoting chronic wound healing. The Smart™ matrix should outperform most other current treatment methods as the Smart™ matrix provides already activated autologous fibroblast cells in the periwound tissue with a conduit of fibronectin domains to infiltrate the hyaluronan scaffold and affect rapid granulation.

The fourth issue, the prevention of scar formation, is addressed by the difference in HA concentration and longevity in scar-forming adult wound healing and in non-scarring fetal tissues. In both adult and fetal animals, hyaluronan concentration becomes highly elevated until around the third day post injury. At that time, the concentrations diverge: adult HA concentration quickly falls, whereas fetal HA remains high as the wound regenerates without scarring (Lorenz et.al., 1993). If a hyaluronan-based Smart™ matrix retains the high HA concentration seen in fetal wounds, perhaps scar formation will be inhibited.

The fifth issue, the ease of application, should be addressed by the acellular nature of the Smart™ matrix. After standard debridement of the wound, the Smart™ matrix can be simply applied as a dry sponge or powder to the wound. The Smart™ matrix can then be easily hydrated through external means, or by absorbing fluid from the wound space. A standard dressing would then be applied to protect the healing wound. Cell-based products, on the other hand, are much more complicated to produce, maintain, and apply.

The sixth issue, the manufacture and distribution of the final product at low cost to the manufacturer as well as to the consumer, is again addressed by the acellular nature of the Smart™ matrix. Whereas cell-based products must be kept at rigorously controlled conditions to protect cell viability, the Smart™ matrix can be manufactured and stored under a wider set of conditions. This influences final costs at the junctures of manufacture, shipping, and application. Whereas cell-based products, even those stored cryogenically, have a limited shelf-life, the Smart™ matrix can be lyophilized for long-term storage. This allows for not only lower costs in regard to wasted unused product, but saves cost in shipping and even production, as larger lots can be manufactured at lower costs per unit.

The thesis encompasses two specific aims. In the first aim, the potential of specific recombinant biologically active domains of human fibronectin to support human fibroblast migration in a two-dimensional assay is explored. This is an important step to establish proof of concept for fibroblast migration on the three functional domains of human fibronectin. Previous work in our lab has shown clear fibroblast migration across functional domains of rat fibronectin (Clark et.al., 2002), but has not shown migration across equivalent domains of human fibronectin. Additionally, the agarose droplet migration assay that was used allows the exploration of fibroblasts on adsorbed fibronectin and its domains. This method avoids using fibronectin as a chemotactic factor. Furthermore, the agarose droplet migration assay allows for the fibroblasts to interact with the fibronectin peptides without interacting with the encapsulating matrix they are in. This is in contrast to previous explorations in collagen migration assays (Greiling and Clark, 1997), where the fibroblasts interacted with, and contracted the collagen.

In the second aim, a three-dimensional hyaluronan scaffold decorated with the biologically active domains of fibronectin is developed. As in the hypothetical Smart™ matrix, the fibronectin fragments will be bound to the HA scaffold. To accomplish this aim, a crosslinking method must be optimized to attain a crosslinked scaffold that facilitates fibroblast infiltration, while retaining the functional activity of the fibronectin fragments that are crosslinked therein.

Previous work in our lab has shown that fibroblasts can migrate within an uncrosslinked 1 % hyaluronan fluid (Greiling et.al., unpublished data). This data shows that hyaluronan has the potential of providing the necessary facilitation of fibroblast migration at this concentration. However, the in vivo wound environment is flush with hyaluronidase, which degrades uncrosslinked hyaluronan very quickly. For the Smart™ matrix to avoid uncontrolled degradation in a chronic wound bed, the hyaluronan must be crosslinked intramolecularly.

Additionally, preliminary work in our lab shows that fibroblasts exhibit robust migration within an uncrosslinked hyaluronan fluid with the addition of intact fibronectin. However, for the Smart™ matrix, it is vital that the functional domains of fibronectin be covalently bound to the scaffold to provide traction for the fibroblast to migrate. Otherwise, the domains would be free-floating in the wound environment, similar to the clipped fragments of the natural state of the chronic wound.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture