Covalently crosslinked chitosan hydrogel formed at neutral pH and body temperature

Water-soluble chitosan having double bonds (CS-MA-LA) was synthesized by sequential grafting of methacrylic acid (MA) and lactic acid (LA) via the reaction between amino groups and carboxyl groups under the catalysis of carbodiimide. Its molecular structure was verified by FTIR and (1)H NMR characterizations. Elemental analysis measured grafting ratios of 19% and 10.33% for MA and LA, respectively. CS-MA-LA was readily soluble in pure water and did not precipitate till pH 9. Gelation of the CS-MA-LA was realized by thermal treatment at body temperature under the initiation of a redox system, ammonium persulfate (APS)/N, N,N',N'-tetramethylethylenediamine (TEMED). The gelation time could be mediated in a wide range, e.g. from 6 to 20 min, by reaction temperature and/or initiator's concentration. 3T3 fibroblast culture showed that the cytotoxicity of the hydrogel extractant was dependent on the cell seeding number and the initiator's concentration. With enough number of cells (>2.5 x 10(4)) and low initiator's concentration (5 mM), the cytotoxicity introduced by the initiator is very minimal and negligible. Although the hydrogel could cause acute inflammation and foreign body reaction, no tissue necrosis and malignant infection were evidenced in vivo, demonstrating that the material has better histocompatibility. These features have endowed the chitosan with great opportunity as injectable biomaterials, which may find wide applications in the rapidly developed fields such as tissue engineering and orthopedics.