The use of physical hydrogels of chitosan for skin regeneration following third-degree burns
Introduction
Every year in the world, hundred thousands patients require hospitalisation for burn injury [1]. The major lesions require a rapid resection of necrotic tissue, and an immediate cover recuperates the most important skin function (physical barrier). The most conventional treatment uses skin grafts. These grafts can compensate the tissue loss at multiple levels acting as occlusive dressings, providing both skin replacement and stimuli for healing [2]. Problems associated with grafts have prompted the research toward an alternative that would be more widely available with properties close to those of a natural skin. Research in this field resulted in the use of biosynthetic materials and tissue engineered living skin replacement. Three types of skin substitutes can be considered: those consisting only in epidermal equivalents, those encompassing dermal components from processed skin and those possessing distinct dermal and epidermal components, referred to as composite skins [3]. Most of skin substitutes consist in matrix seeded cell cultures. Because of Rheinwald and Green's works [4] cultured epidermal grafts were developed. They proved that a combination of growth factors and irradiated murine 3T3 fibroblasts was likely to support the proliferation of human keratinocytes on synthetic polymer surfaces. Keratinocyte sheets were formed, deposited on gauze and grafted on wounds. Different supports for these sheets have been tested such as polyurethane [5] and derivatives of hyaluronic acid [6]. These systems did not yield satisfactory results due to their mechanical instability. They were brittle and then, a blistering of the epidermis occurred. The grafted skin was also reported to be more prone to shrinking yielding poor aesthetic results [7].
Although numerous studies on dermal equivalents used as grafts were reported, acellular matrices are still proposed. We may mention the case of: Alloderm™, obtained from cadaver skin [8], Integra™, constituted of glycosaminoglycans and bovine collagen, covered with a silicon sheet [9]. Cellular dermis equivalents like Dermagraft™ consisting in a culture of neonatal fibroblasts onto a bioresorbable synthetic matrix are also considered [10]. Composites with both dermal and epidermal components were first introduced by Bell et al. but were not fully satisfying for the treatment of severely burned patients [11]. Nevertheless, they allowed the development of new strategies and new materials such as Apligraft® [12] constituted of a fibroblast/collagen matrix on which a fine layer of stratified human epithelium was cultivated. Biodegradable polymeric three-dimensional matrices are gaining attention for cell culture such as sponges of collagen/polycaprolactone [13]. Damour et al. [14] developed a sponge constituted of bovine type I collagen/chondroitin-4-6 sulphate/chitosan for in vivo skin reconstruction.
In this paper, we propose a new acellular biomaterial based on physical hydrogels of chitosan to cover burn areas. The major goal is to achieve a permanent skin regeneration with both dermal and epidermal tissues, good functional and aesthetic characteristics. Chitosan, fully absent in mammals corresponds to an interesting series of natural glycosaminoglycans possessing the rare property of bioactivity [15]. Chitin with chitosan constitute the series of the linear copolymers of (1→4)-2-amino-2-deoxy-β-d-glucan (GLcN) and (1→4)-2-acetamido-2-deoxy-β-d-glucan (GlcNAc). DA, the degree of acetylation refers to the molar fraction of N-acetyl units present in the polymer chains. When the distribution of the two constitutive residues is random, chitosan corresponds to DAs below 70% [15], and is soluble in dilute acidic solutions. Chitosan-based matrices have been widely used in the biomedical field: for cells encapsulation [16], drug delivery [17], cell culture [18], hyaline cartilage repair [19] and bone reconstruction [20]. In the case of wound healing, Ueno et al. [21] demonstrated that chitosan in the form of a chitosan-cotton blend was an accelerator of wound healing by the activation and infiltration of polymorphonuclear cells at the wound site. Recently, Mizuno et al. also concluded that chitosan was a good wound healing material and that incorporation of basic fibroblast growth factors in the chitosan material accelerated the rate of healing [22]. Park et al. [23] studied in vitro the effect of carboxymethylchitosan on the proliferation of normal human skin and keloid fibroblasts. In vitro studies also showed that chitosan accelerated the proliferation of keratinocytes [24].
Pure physical hydrogels of chitosan were recently obtained [25], [26], [27]. These materials only use intermolecular physical cross-links of low energy (⩽ kT): hydrogen bonding and hydrophobic interactions to elaborate a three-dimensional network of polymer chains, without any use of external chemical agent. Encouraging results were obtained with chondrocyte cultures [19]. In this work, we prepared a bi-layer physical hydrogel, only constituted of chitosan and water. This material was tested in vivo for the skin reconstruction after third-degree burns on pig back skins. A kinetics study of the healing showed the full skin reconstruction. The present data also allowed us to confirm the first results obtained with these gels by Montembault et al. [19] for in vitro chondrocyte cultures inducing the production of a cartilaginous matrix.
Section snippets
Purification of chitosan
We used a chitosan produced from squid pens, purchased by Mahtani Chitosan (batch number 114, 15/11/02). For purification, the polymer was dissolved at 0.5% (w/w) in acetic acid to achieve the stoechiometric protonation of the NH2 sites. The mixture was filtered successively on 3, 1.2, 0.8 and 0.45 μm Millipore membranes. Then, the polymer was precipitated by means of concentrated ammonia (28% (w/w)). After several washings in deionised and distilled water and centrifugation steps until a
Experimental thermal injury
About 30 min before starting the procedure, animals were sedated with Vetranquil™ (acepromazine) 0.1 mL/kg by means of an intra-muscular injection. Anaesthesia was performed using Imalgene-Rompun™ (10 mg/kg ketamine+2 mg/kg xylazine) by means of an intra-muscular injection completed with a gaseous procedure (O2, 0.9 L/min plus Isoflurane™). Under anaesthesia, 5×5 cm2 full-thickness dorsal burns were processed on each pig by applying during 30 s a cubic copper piece heated at 100 °C. A manometer
Formation of bi-layer physical hydrogels
In order to partly mimic the skin, we developed physical hydrogels of chitosan, constituted of two physically linked layers of different mechanical property and density. The literature only reports on multilayer hydrogels, which different layers are chemically grafted together [31], [32]. Whatever the DA, a chitosan physical hydrogel can be formed [26], provided three major conditions are observed: (1) the initial polymer concentration must be over C*, the critical concentration of chain
Conclusion
A new biomaterial consisting in a bi-layer physical hydrogel of chitosan without any external cross-linking agent was elaborated and tested for third-degree burns treatment. The results reported in this work were obtained with a pig dorsal deep burn model. From our observations, we may consider that our bi-layer physical hydrogel is able to induce an appropriate response for the reconstruction of the skin after a third-degree burn injury, on a limited area. It represents both an ideal material
Acknowledgements
This work was funded by a grant from the French “Délégation Générale pour l’Armement” (DGA). We thank Sylviane Guerret (Novotec, Lyon, France) for histological and immuno-staining analyses.
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