Morphology and gelation of thermosensitive chitosan hydrogels
Introduction
Hydrogels are an ideal class of polymeric material for biomedical applications. They contain only small amounts of polymeric material (typically hydrogels are in the range 1–30 wt.% in aqueous solvent), have low interfacial tension, high molecular and oxygen permeability and mechanical properties that resemble physiological soft tissue [1]. Hydrogels are used for cell encapsulation [2], [3], [4], [5], lubrication and cushioning of joints [6], [7], drug delivery [8], [9], [10], [11], [12], [13] or tissue-engineered scaffolds [13], [14], [15], [16]. Drug release profiles, the flow of nutrients to seeded cells and enzymatic degradation are all diffusion-controlled, which is determined by interconnected porosity and volume of water phase present. The shape, size, and size distribution of pores are also important parameters for the seeding of cells within the hydrogel. To fully exploit a hydrogel, these morphological characteristics must be known and controlled or tailored for the intended application.
Many techniques are available for microstructural analysis of macro-porous hydrogels, however most raise issues such as collapse of pore structure during dehydration [16], [17], [18] and lack of contrast and resolution. Laser scanning confocal microscopy (LSCM) bypasses these issues by imaging hydrogel morphology in the native hydrated state and has a resolution of 0.35 μm (for a more comprehensive description see Srinivasarao [19]). Conjugation of a water-soluble fluorochrome to the material to provide contrast is important. The conjugated fluorophore is assumed to have minimal effect on the behaviour of the hydrogel [20], because the hydrogel molecules are thousands of times larger and more concentrated than the fluorophore (e.g., in this instance, the molar ratio of chitosan amine to fluorescein isothiocyanate (FITC) is 66,000:1).
Chitosan is a polysaccharide hydrogel. It is composed of (1,4)-linked 2-amino-2-deoxy-β-d-glucan (Fig. 1), produced by deacetylating chitin, with applications ranging from biomedical to cosmetic. The properties of chitosan are primarily governed by the degree of deacetylation (DD, determined from the relative amounts of acetyl and amine groups at the C2 position, labelled R in Fig. 1).
Chitosan is soluble in dilute acidic solutions, but phase-separates at pH > 6 to form a hydrogel. However, on addition of glycerophosphate salt (GP) to a chitosan solution, the pH can be raised to neutral without causing phase-separation [21], [22]. The system becomes thermally sensitive, forming a gel above a certain temperature. To date, the only study of chitosan/GP morphology known to the authors was conducted after freeze-drying, by SEM [9]. This study aims to examine the morphology of chitosan/GP, in its native, hydrated state, and to gain a better understanding of the gelation mechanism.
Section snippets
Materials
Chitosan (Sigma) was purified by dissolving in 0.1 M HCl (BDH), filtering through grade 3 filter paper (Whatman), heating, and then when cooled, stirring with granulated carbon and refiltering. The chitosan was precipitated by adding 100 mL chitosan solution drop wise to 600 mL 0.1 M KOH (Aldrich). The precipitate was collected, rinsed twice with distilled deionised water, and freeze-dried for 48 h. β-Glycerophosphate disodium salt (Sigma) was used as received, while the fluorophore FITC
Scanning electron microscopy
The morphology of the dry chitosan/GP gels was initially examined by SEM. Fig. 2 shows sample SEM images of two concentrations, of which Fig. 2A is typical. It shows a very fibrous microstructure, with large interconnected areas between fibres.
Fig. 2B, taken at a fracture surface to reveal the bulk microstructure, shows that during water loss much of the pore structure is compacted. At the surface, however, a different effect is seen. While the fibrous structure exists, a skin layer has formed (
Conclusion
Chitosan/GP is a low composition thermosensitive hydrogel, and hence examination of its morphology is difficult. This study shows that LSCM is a valuable technique for imaging hydrogel microstructure, giving both qualitative and quantitative data. The microstructure of dehydrated chitosan/GP is very different from the gel, and composition was found to play a role in determining the microstructure. The images of chitosan/GP by LSCM show a heterogeneous microstructure and suggest that the kinetic
Acknowledgements
We would like to thank W. Cook for use of the UV–vis spectrophotometer, and I. Harper, M. Kitchen, and J. Gillam for useful discussions on LSCM data analysis. This project was funded by an Australian Postgraduate Award, the CRC for Polymers and ARC project DP0450618.
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