Disulfide-Crosslinked Chitosan-Gelatin Hydrogels

Many of hydrogels derived from the natural polymers have been adapted for tissue engineering materials with proper modification. In this work, Thiolated chitosan and gelatin were synthesized by using thioglycolic acid (TGA), 4-butyrothiolactone (BTA) or 3,3'-dithiobis(propionic hydrazide) (DTPH). Mediated by carbodiimide (EDAC) or imidazole, mercapto group were covalently linked to chitosan and gelatin. Dependent on the pH-value and modifier, the thiolated polymer derivatives displayed different mercapto group contents. At a pH-value of 3, the chitosan-thiomers reached its maximum mercapto group contents for about 100μmole/g polymer. Furthermore, mercapto group contents of thiolated gelatin synthesized by DTPH method are more larger than that of synthesized by BTA, because of the high reactivity between hydrazide and carboxyl groups compared to amino and carboxyl ones.A new disulfide cross-linking method was developed for the preparation of blended chitosan-gelatin hydrogels to form a synthetic, covalently linked mimic of the extracellular matrix (ECM). Solutions of thiolated chitosan and thiolated gelatin in varying blends were prepared and cross-linked by disulfide bond formation in air. Hydrogel films were dried and further cross-linked with dilute hydrogen peroxide.The swelling ratio and degradation rate of disulfide cross-linked chitosan-gelatin hydrogels were strongly depended on compositions and cross-linking degree. The cross-linked films were degraded more rapidly by collagenase compared to lysozyme and only a little weight loss was observed in enzyme-free buffer. Cytotoxicity evaluation using L929 fibroblasts as model system showed that the chitosan-gelatin hydrogels were nontoxic. Fluorescent photomicrographs showed good morphology of L929 in these hydrogel films. MTT assay confirmed that L929 proliferation on the hydrogel surface was comparable to that on control (TCPS). The macroporous chitosan-gelatin hydrogel scaffolds were also fabricated through freeze-dry technique. The porosity and mean diameter of scaffold were determined by the concentration of the solution and pre-freezing temperature, respectively. The hydrogel scaffolds showed a network of uniform, interconnected pores with size in the range of 75-250μm.