Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering

Abstract

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.

Introduction

The skin loss is one of the oldest and still not totally resolved problems in surgical field. Due to the spontaneous healing of the dermal defects would not occur, the scar formation for the full thickness skin loss would be inevitable unless some skin substitutes are used. In the past decades, many skin substitutes such as xenografts, allografts and autografts have been employed for wound healing. However, because of the antigenicity or the limitation of donor sites, the skin substitutes mentioned above cannot accomplish the purpose of the skin recovery and yet not be used widely [1], [2], [3], [4], [5]. Therefore, many studies are turning toward the tissue engineering approach, which utilizes both engineering and life science discipline to promote organ or tissue regeneration and to sustain, recover their functions [6], [7], [8], [9]. One crucial factor in skin tissue engineering is the construction of a scaffold. A three-dimensional scaffold provides an extra cellular matrix analog which functions as a necessary template for host infiltration and a physical support to guide the differentiation and proliferation of cells into the targeted functional tissue or organ [10], [11]. An ideal scaffold used for skin tissue engineering should possess the characteristics of excellent biocompatibility, suitable microstructure such as 100–200 μm mean pore size and porosity above 90%, controllable biodegradability and suitable mechanical property [12], [13], [14], [15]. Collagen is known to be the most promising materials and have been found diverse applications in tissue engineering for their excellent biocompatibility and biodegradability. However, the fast biodegrading rate and the low mechanical strength of the untreated collagen scaffold are the crucial problems that limit the further use of this material. Cross-linking of the collagen-based scaffolds is an effective method to modify the biodegrading rate and to optimize the mechanical property.

For this reason, the cross-linking treatment to collagen has become one of the most important issues for the collagen-based scaffolds. Currently, there are two different kinds of cross-linking methods employed in improving the properties of the collagen-based scaffolds: chemical methods and physical methods. The latter include the use of photooxidation, dehydrothermal treatments (DHT) and ultraviolet irradiation, which could avoid introducing potential cytotoxic chemical residuals and sustain the excellent biocompatibility of the collagen materials [16]. However, most of the physical treatments cannot yield high enough cross-linking degree to satisfy the demand of skin tissue engineering. Therefore, the treatments by chemical methods are still necessary in almost all cases. The reagents used in the cross-linking treatment recently involve traditional glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), polyglycidyl ether and polyepoxidic resins, etc. [17], [18], [19], [20], [21]. GA is a kind of bifunctional cross-linking reagents that can bridge amino groups between two adjacent polypeptide chains and has become the predominant choice in skin tissue engineering because of its water solubility, high cross-linking efficiency and low cost [22].

Chitosan is another biomaterials used in a variety of biomedical fields such as drug delivery carriers, surgical thread, and wound healing materials [23]. Due to its many advantages for wound healing such as hemostasis, accelerating the tissue regeneration and the fibroblast synthesis of collagen, many applications of chitosan in skin tissue engineering have been reported [24], [25], [26], [27]. In addition, chitosan can function as a bridge to increase the cross-linking efficiency of GA in the collagen-based scaffolds owing to the large number of amino groups in its molecular chain (Fig. 1). Hence, one can expect that less GA could be used in the presence of chitosan and the potential cytotoxicity of GA might be decreased.

Herein we describe the fabrication of collagen porous scaffold in the presence of 10 wt% chitosan, which functions as a cross-linking bridge in the further treatment of GA cross-linkage. The microstructure, the swelling capacity, as well as the degradability both in vivo and in vitro of the collagen/chitosan scaffold were investigated. In vitro culture of human dermal fibroblasts and in vivo animal tests demonstrated that the scaffolds showed good cytocompatibility and could effectively guide the infiltration and growth of fibroblasts.