Formation of Chitosan-Based Hydrogels Network - Chitosan Based Hydrogels: Functions and Applications - page 189

The biocompatibility of the chitosan network is the final evaluation index and it is a
comprehensive effect of the above physical-chemical properties. Above all, different
chemical structures and properties of the network result from different cross-linkers and
cross-linking mechanisms. Cross-linkers are molecules with at least two reactive func-
tional groups that allow the formation of bridges among polymeric chains. To date, the
most commonly used cross-linkers with chitosan-based biomaterials are dialdehydes
(such as glyoxal and in particular glutaraldehyde (GA), genipin, diepoxide, EDC, etc.). The
details of the cross-linking mechanism and properties of the chitosan-based cross-linking
network will be provided below.
4.2.3.1 Dialdehyde-Cross-Linked CS
GA is a commonly used cross-linker for chitosan and the cross-linking mechanism is
shown in Figure 4.6 [93]. The chitosan-based biomaterial networks cross-linked by GA
have many advantages. In fact, the easiness of synthesis, the speed of reaction, the mild
experimental conditions (room temperature, pH around 4.5), and the acceptable biocom-
patibility of the reactants make these networks particularly promising for the synthesis of
biomaterials in situ [94,95]. The reaction between amino groups of chitosan and GA is
highly unlikely to proceed homogeneously, and the absolute stoichiometry of the reaction
cannot be determined by the [-CHO]/[-NH 2 ] values. The amount of adsorbed proteins on
the GA-cross-linked chitosan (GCCS) surface decreases because the positive-charged
amino groups of chitosan are cross-linked by the GA. However, there are some aldehyde
groups on the surface of GCCS that may bind proteins via their amino groups by the for-
mation of azomethine bonds [96]. The mechanical performance of chitosan films or fibers
can be improved by using GA as a cross-linking agent [97]. Chitosan cross-linked by GA
and spacer group glycine has been reported by Gupta and Kumar [98]. The cross-linking
network can be hydrolyzed in an acidic medium due to a higher swelling degree than at
basic pH. These preliminary results suggest the possibility to obtain a desired pH-sensi-
tive drug delivery system using GCCS as a carrier.
A chitosan film is formed by solution casting and then cross-linked by immersion in a
GA aqueous solution. The residual aldehyde groups are blocked with a glycine solution.
Neutralizing and washing until the film pH returns to the physiological range, primary
rat heptocytes are seeded on the film. Hepatocytes sparsely adhere to the chitosan film in
culture media. Results indicate that chitosan is a poor substrate for hepatocytes' attach-
ment. However, it is suitable for sustaining hepatocytic functions [99].
On the other hand, GA is neurotoxic, and glyoxal is mutagenic. Therefore, even if prod-
ucts are purified before administration, the presence of free unreacted dialdehydes in the
products cannot be completely excluded.
4.2.3.2 Genipin-Cross-Linked Chitosan
Genipin is a natural cross-linking reagent which has recently been used as a cross-linking
agent for chitosan and proteins containing residues with primary amine groups. It is not
cytotoxic in vitro [100] and is biocompatible after injection in rats [101]. The cross-linking
mechanism consists of two reactions, involving different sites on the genipin molecule (as
shown in Figure 4.7) [102,103]. The first step is the nucleophilic attack of amino group of the
genipin C-3 carbon atom from a primary amine group to form an intermediate aldehyde
group. The newly formed secondary amine reacts with the aldehyde group to form a
heterocyclic compound. The step that follows is a nucleophilic substitution reaction that