Morphology and gelation of thermosensitive chitosan hydrogels

Abstract

The morphology of physical hydrogels is often difficult to examine due to the delicate nature of the system and therefore has not been studied in detail. Chitosan/GP (glycerophosphate salt) is a significant hydrogel in the biomedical and cosmetic fields as it is thermosensitive and contains less than 5% polysaccharide. The morphology of this system was examined with laser scanning confocal microscopy (LSCM) to image the gel morphology. The images indicate that the gel is quite heterogeneous, and power spectra reveal a fractal-like morphology. A study of composition found that increasing chitosan concentration increased the amount of polymer-rich phase present in the gel, and that the smallest aggregates decreased in size.

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.