Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing

Abstract

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm™ or Tegaderm™ alone. A significant increase in re-epithelialization was observed on day 5 (p<0.001) and day 7 (p<0.05) for wounds treated with a GAG film plus Tegaderm™ versus those treated with Tegaderm™ alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components, presentation of growth and differentiation factors, and cell migration can readily occur.

Introduction

Glycosaminoglycans (GAGs), including hyaluronan (HA) and chondroitin sulfate (CS), are aminosugar-containing polysaccharides in the extracellular matrix (ECM) of all vertebrates. HA is the only non-sulfated GAG and is comprised of alternating units of β-1,4-linked d-glucuronic acid and (β-1,3) N-acetyl-d-glucosamine. HA is non-immunogenic and forms highly viscous aqueous solutions, endowing HA with unique physicochemical properties as well as distinctive biological functions [1]. HA has been implicated in the preservation of tissue hydration, in the regulation of permeability of other substances by steric exclusion phenomena, and in the lubrication of joints [2]. HA plays an important role in the structure and organization of ECM, including the maintenance of extracellular space and the transport of ion solutes and nutrients. HA also binds specifically to proteins in the ECM, on cell-surface receptors, and within the cell cytosol. These protein–ligand interactions stabilize the cartilage ECM [2], [3], regulate cell adhesion and motility [4], [5], and mediate cell proliferation and differentiation [6]. HA signaling occurs during morphogenesis and embryonic development [7], modulation of inflammation [8], and in the stimulation of angiogenesis and healing [9].

Another member of the GAG family is CS, which is comprised of alternating units of β-1,3-linked glucuronic acid and (β-1,4) N-acetyl-galactosamine (GalNAc) and is sulfated on either the 4- or 6- position of the GalNAc residues. The 4-sulfate (CS-A) and 6-sulfate (CS-C) generally occur in covalent linkage to a core protein, thus producing a proteoglycan. Aggregan is the main proteoglycan in cartilage, and its primary function is to swell and hydrate the collagen fibril framework. Versican, another CS proteoglycan, is involved in intracellular signaling, cell recognition, and connecting ECM components to cell-surface glycoproteins [10]. Additionally, CS proteoglycans, such as neurocan and phosphacan, provide cues for cell orientation during axon growth and pathfinding [11].

Wound healing is a complex and orderly sequence of events that involves a variety of cell-types and both extracellular and intracellular signals. Surprisingly, the exact role of GAG molecules in the wound healing process remains unresolved. It appears that tissue concentrations of HA increase in early granulation tissue and then decrease in the later phases of wound healing, when the CS proteoglycan concentrations are increasing [12]. The presence of HA is believed to provide a matrix that is more readily penetrated by cells, and thus high concentrations of HA are correlated at times of increased cell movement and proliferation. The importance of HA and HA receptors in cell motility and differentiation is consistent with this interpretation [6]. Eventually, in most tissues, HA is degraded by hyaluronidase (HAse) and is replaced with proteoglycans to provide the tissue with more resilience.

The role of GAG molecules is even more interesting and complex in fetal wound healing. After injury, HA levels in both fetal and adult wounds are elevated. The fetal HA levels remain elevated and are associated with scarless repair [13]. Additionally, human fetal skin is structurally different from adult skin in both its distribution and composition of HA and large, aggregating CS proteoglycans [14]. Fetal skin contains sparse amounts of fibrillar collagen embedded in a highly hydrated amorphous matrix composed principally of HA and sulfated proteoglycans, creating a matrix that supports active proliferation, migration, and differentiation events, which are required for developing tissues [15].

Recently, GAG molecules have been chemically modified [16], [17], [18], [19], creating new bio-materials with important bio-medical applications [20], [21]. Chemical modification allows their physicochemical and mechanical properties to be tailored, while retaining their natural bio-compatibility [20], bio-degradability, and lack of immunogenicity. Application of such new hydrogel bio-materials [22] include scaffolds for tissue engineering, materials for localized drug delivery, barriers to prevent post-surgical adhesions, and dressings and matrices for wound healing and bone repair. By creating a synthetic ECM, dressings prepared from GAG-derived hydrogel films should participate actively in the wound healing process rather than simply being passive barriers to desiccation. We describe herein the preparation and characterization of two new pliable GAG hydrogel films, and we present data for the use of these films in the healing of full-thickness wounds.