Development of collagen-EDC scaffolds for skin tissue engineering: physicochemical and biological characterization
Abstract
A leading consequence of burns is the loss of large extensions of skin. Thus, skin tissue engineering has been increased and promoted development of biomimetic skin scaffolds. Type I collagen is one of the most materials used in tissue engineering due to its biological characteristics. However, the applications of collagen as biomaterial are severely limited by its reduced physicochemical and mechanical properties, such as high susceptibility to enzymatic degradation in vivo and low thermo stability. To enhance collagen properties, crosslinked collagen scaffolds at different concentrations of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were prepared by freeze-drying technique. The effect of crosslinking and concentration on scaffolds physicochemical and biological behavior was evaluated. Scaffolds morphology was observed by Scanning Electron Microscopy, showing in all cases an appropriate microstructure for biological applications. Differential Scanning Calorimetric showed an increase in shrinkage temperature (TS) with increase in EDC concentration. Infrared Spectroscopy suggested that the secondary structure of collagen is not affected after the crosslinking. Enzymatic degradation test indicated that scaffolds treated with EDC dissolved slowly in enzymatic solution (just 12% of degradation after 96 h). Cell viability and attachment tests suggested that EDC treatment do not affect the excellent biological characteristics of collagen.
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Introduction
Skin is the largest organ of the body and it is composed of epidermis, dermis, and hypodermis layers, together with a complex nerve and blood supply systems [1], [2]. Loss of large part of skin, related to illness or injury, would not only affect the appearance of the patient, but also can lead to infection and even causing death. Thus, the necessity of skin substitutes for wound healing has increased skin tissue engineering research and promoted development of biomimetic skin scaffolds that help to regenerate large points of damaged skin [3], [4].
Collagen has been widely used to fabricate scaffolds due to its high biocompatibility, low antigenic response and because it naturally contains cell adhesion motives that improve cell-scaffold interactions [5]–[8]. Type I collagen is the most common type of collagen and it is the major protein of all connective tissue, such as bone, tendon, cartilage and skin [9]–[12]. Despite the huge efforts and developments, the uses of collagen for tissue engineering applications is currently limited by its high susceptibility to enzymatic degradation and low thermo stability in vivo [13]–[15]. For the development of new skin tissue engineering therapies, the enhancement of thermal, mechanical and enzymatic stability of collagen is needed. The crosslinking is the most used method collagen properties [8], [16], [17].
Glutaraldehyde has been extensively used as crosslinker, but it is associated with cytotoxicity effects, reduced cellular ingrowth in vitro and in vivo [13], [17], [18]. The search of “green” crosslinkers that avoid denaturation protein and can be easily removed after the crosslinking is the topic of current research in the field. Among potential alternatives, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) has demonstrated to be a mild[8], [19] and water-soluble crosslinker. EDC induce the formation of covalent amine bonds thought carboxyl and amino groups of collagen[8], [19], [20]. Usha et.al.[19] reported the physicochemical behavior of collagen crosslinked with EDC (10 mM). However, they do not report the biological performance of the scaffolds obtained.
Conclusion
Type I collagen scaffolds were successfully stabilized by crosslinking treatment with EDC. The results of the physicochemical characterization of the crosslinked scaffolds indicate that EDC treatment has not significantly altered the porous morphology of scaffolds, but improve the structural stability and thermal properties, necessary behavior in materials for tissue engineering applications. Scaffolds showed the same proliferation profile and before and after crosslinking treatment, besides the cells showed affinity for the crosslinked scaffolds. All these results suggest that this study could be useful for development a collagen-based biomaterial for tissue engineering applications having suitable physicochemical properties and reduced antigenicity, even with high EDC concentrations. The findings of this study are limited to the in vitro behavior. Furthermore, they are a starting point and the in vivo performance should be evaluated.