Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering
Introduction
A tissue engineering approach to the regeneration of new tissue involves the use of cells seeded on a scaffold. Chitosan has potential as a biomaterial for tissue engineering scaffolds because it is biodegradable, easily formed into structures under mild processing conditions and can be chemically modified. The scaffold provides a necessary template and physical support to guide the differentiation and proliferation of cells into targeted functional tissues or organs [1]. The scaffold should be reproducibly processable into three-dimensional structures [2]. It should absorb body fluid for transfer of cell nutrients and metabolites through the material [3]. To avoid exudate accumulation or excessive dehydration, an ideal dressing or graft would control the loss of water from the skin at an optimal rate [4], [5], [6].
Transport issues such as nutrient delivery, waste removal, protein transport, gaseous exchange, and general vascularization and guided tissue regeneration are governed by the pore structure of the scaffold [7]. Pore sizes recommended for skin scaffold ranges are <160 μm, 15–100 μm [3] and 100–200 μm [9] with a desired porosity of 90% to provide sufficient space and surface for cell seeding into temporary scaffold prior to implantation [1].
A tissue engineering scaffold should be biocompatible and biodegradable, and the degradation product should be non-toxic. A degradation time of 25 days is suitable for healing acute wounds (burn and skin excision) [4] or about 8 weeks for chronic wounds (diabetic ulcer, pressure ulcer) [8]. Scaffolds can be developed using a variety of materials including synthetic or natural polymers [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Among these materials, chitosan, the deacetylated derivative of chitin, is a promising candidate; it is biodegradable in the presence of lysozyme, an enzyme present in the human body and also produced by macrophages during wound healing. Degradation by lysozyme permits resorption of the scaffold and liberation of N-acetyl glucosamine, glucosamine and other oligomers which activate macrophages and are incorporated into the extracellular matrix for the rebuilding of physiological normal tissues. However, chitosan has low mechanical integrity and degrades rapidly especially in an acid medium and the presence of lysozyme [14]. Glutaraldehyde (GTA) is widely used to improve the structural properties of chitosan and other polymers by crosslinking the amino groups [16]; however, GTA is generally considered to be toxic [17] and alternative crosslinkers are sought [15]. Dimethyl 3,3,dithio bis propionimidate (DTBP) is a potential alternative crosslinking agent, that was successfully used to crosslink collagen [18], [19]. The resulting collagen scaffolds had improved mechanical properties and a slower degradation rate compared to an uncrosslinked collagen. They had a rate of degradation similar to that of GTA-crosslinked collagen. In vivo experiments found DTBP-crosslinked collagen to be more biocompatible than GTA-crosslinked collagen, when implanted into the backs of rats [18].
The objective of this research was to develop three-dimensional scaffolds from chitosan crosslinked with DTBP (shown in Fig. 1) and to evaluate the tissue engineering properties of these matrices with respect to uncrosslinked and GTA-crosslinked chitosan matrices. Additionally, the effect of the degree of deacetylation (DDA) of chitosan on the tissue engineering properties of the scaffolds was evaluated.
Section snippets
Materials
Chitosan with a specified DDA (80%, 90% and 100%) was obtained from Carbomer Inc. Lysozyme from chicken egg white (58,100 units/mg), DTBP dihydrochloride and GTA grade 11 and 25% aqueous solution were obtained from SIGMA ALDRICH. All other chemicals were obtained from SIGMA ALDRICH.
Preparation of chitosan scaffolds
Five ml of 1.0%, 1.5% or 2.0% w/v chitosan solution (in 3.0% acetic acid) was transferred to a mold of diameter 33 mm and height 5 mm, frozen at −20 °C for 24 h, and then freeze dried for 24 h. The resulting samples were
Results
Freeze drying resulted in porous chitosan scaffolds, as shown by the SEM images in Fig. 2. Uniformly sized scaffolds were reproducible formed; CH, CH–DTBP and CH–GTA scaffolds had average sizes of: diameter (mm)=3.19±0.05, 2.96±0.046, 3.17±0.064 and thickness (mm)=0.461±0.04, 0.43±0.034, 0.385±0.028, respectively. The crosslinking density of chitosan scaffolds crosslinked with 0.1, 0.3 and 0.5 w/v% DTBP are shown in Fig. 3. Crosslinking density increases with increasing DTBP concentration.
Discussion
DTBP was used as an effective crosslinker for chitosan, resulting in a scaffold with similar physical properties to GTA-crosslinked chitosan scaffolds. Diimidoesters such as DTBP form crosslinks between free amino groups separated by a distance equivalent to its molecular length. GTA on the other hand, can polymerize and form crosslinks of various lengths possibly linking residues that are spaced farther apart. However the presence of disulfide linkages in the DTBP-crosslinked scaffolds may be
Conclusion
This paper describes the results of the first studies using DTBP to crosslink chitosan. The present study indicates that porous scaffolds for tissue engineering applications with physical properties comparable to GTA can be fabricated using DTBP as a crosslinker for chitosan. The toxicity of DTBP-crosslinked chitosan scaffolds is improved over GTA-crosslinked scaffolds. The scaffold properties generally improve with increasing DDA. However a 100% DDA results in effectively zero degradation. The
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