Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing
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.
Section snippets
Materials
Fermentation-derived HA (HA, sodium salt, Mw=1.5×106 Da) was provided by Clear Solutions Biotech, Inc. (Stony Brook, NY). 1-Ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDCI) and adipic dihydrazide (ADH) were from Aldrich Chemical Co. (Milwaukee, WI). Chondroitin sulfate C was obtained from Sigma (St. Louis, MO). Poly(ethylene glycol)-propiondialdehyde (PEG-diald) (Mw=3400 Da) was from Shearwater Polymers, Inc. (Huntsville, AL). Balb/c mice were purchased from Charles River Laboratories
Results
The bio-compatible hydrogel films were prepared using the ADH derivatives of each GAG, in which a pendant hydrazide functionality permits generation of a gel using a small molecule [18] or macromolecular [17] crosslinkers (Fig. 1). Prior to crosslinking, the degree of substitution was determined by integration of the ADH methylene signals using the N-acetyl methyl resonances (δ=1.95∼2.00 ppm) as an internal standard [18]. The degree of ADH substitution of the HA-ADH used in this study was 55%,
Discussion
An ideal wound dressing should have several key attributes. The dressing should protect the wound from bacterial infection, control evaporative water loss and prevent dehydration, control permeability of oxygen and carbon dioxide, absorb wound exudate, and enhance the healing. Additionally, it should be composed of materials that are non-toxic, non-immunogenic, flexible, durable, and comfortable when worn. The search for an “ideal” wound dressing has been long and exhaustive. Synthetic
Conclusion
Herein, the effects of two new GAG hydrogel films on wound healing were evaluated using a mouse full-thickness wound model. Chemically-modified HA and CS were each crosslinked with PEG-diald under bio-compatible conditions to form flexible, hydratable hydrogel films. The films were then evaluated for their wound healing capabilities. Full-thickness wounds were dressed with a GAG film plus Tegaderm™ or Tegaderm™ alone. A significant difference in re-epithelialization was found on days 5 and 7
Acknowledgements
Financial support for this work was provided by Center for Bio-polymers at Interfaces (The University of Utah), by start-up funds provided by The University of Utah to G.D.P., and by a tissue engineering consortium grant from the National Institutes of Health (DC 04336) to Dr. Steven D. Gray. We thank Dr. Ernst Eichwald for valuable discussions and experimental assistance.
References (53)
- et al.
Rapid hyaluronan uptake is associated with enhanced motilityimplications for an intracellular mode of action
FEBS Lett
(1998) - et al.
Hyaluronic acid induces tumour necrosis factor-α production by human macrophages in vitro
British J Plastic Surg
(1997) - et al.
Developmentally programmed expression of hyaluronan in human skin and its appendages
J Invest Dermatol
(1997) - et al.
Cross-linked hyaluronic acid hydrogel filmsnew biomaterials for drug delivery
J Controlled Rel
(2000) - et al.
Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury
Burns
(1998) - et al.
Possibility of wound dressing using poly(l-leucine)/poly(ethylene glycol)/poly(l-leucine) triblock copolymer
Biomaterials
(2000) - et al.
Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes
Biomaterials
(1997) - et al.
Study on gelatin-containing artificial skinI. Preparation and characteristics of novel gelatin-alginate sponge
Biomaterials
(1999) - et al.
Application of benzyl hyaluronate membranes as potential wound dressingsevaluation of water vapour and gas permeabilities
Biomaterials
(1996) - et al.
A new model for studying differentiation and growth of epidermal cultures on hyaluronan-based carrier
Biomaterials
(1999)
Semisynthetic resorbable materials from hyaluronan esterification
Biomaterials
Inhibition of wound contraction with locally injected lathyrogenic drugs
Am J Surg
Functions of hyaluronan
Ann Rheum Dis
Hyaluronanits nature, distribution, functions and turnover
J Int Med
An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development
J Histochem Cytochem
Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility
J Cell Biol
Receptorsregulators of signalling to the cytoskeleton
J Cell Biochem
Hyaluronan in morphogenesis
J Int Med
Dynamic role of hyaluronan (HYA) in connective tissue activation and inflammation
J Int Med
Chondroitin sulfate proteoglycans as mediators of axon growth and pathfinding
Cell Tissue Res
Wound repairoverview and general considerations
Repeated additions of hyaluronan alters granulation tissue deposition in sponge implants in mice
Wound Repair Regen
Versican in human fetal skin development
Anat Embryol
Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate
Bioconjugate Chem
Novel hydrogels of hyaluronic acidsynthesis, surface morphology, and solid-state NMR
J Am Chem Soc
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