Elsevier

Biomaterials

Volume 26, Issue 35, December 2005, Pages 7241-7250
Biomaterials

Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering

https://doi.org/10.1016/j.biomaterials.2005.05.043Get rights and content

Abstract

Chitosan, the deacetylated derivative of chitin, is a promising scaffold material for skin tissue engineering applications. It is biocompatible and biodegradable, and the degradation products are resorbable. However, the rapid degradation of chitosan and its low mechanical strength are concerns that may limit its use. In this study, chitosan with 80%, 90% and 100% degree of deacetylation (DDA) was crosslinked with dimethyl 3-3, dithio bis’ propionimidate (DTBP) and compared to uncrosslinked scaffolds. The scaffolds were characterized with respect to important tissue engineering properties.

The tensile strength of scaffolds made from 100% DDA chitosan was significantly higher than for scaffolds made from 80% and 90% DDA chitosan. Crosslinking of scaffolds with DTBP increased the tensile strength. Crosslinking with DTBP had no significant effect on water vapour transmission rate (WVTR) or water absorption but had significant effect on the pore size and porosity of the samples. All samples showed a WVTR and pore size distribution suitable for skin tissue engineering; however, the water absorption and porosity were lower than the optimal values for skin tissue engineering. The biodegradation rate of scaffolds crosslinked with DTBP and glutaraldehyde (GTA) were reduced while no significant effect was observed in biodegradation of the samples made from 100% DDA chitosan whether crosslinked or uncrosslinked after 24 days of degradation.

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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