The effectiveness of four different reagents, glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), poly(ethyelene glycol) diglycidyl ether (EX 810), and divinyl sulfone (DVS) as crosslinkers for cast hyaluronic acid (HA) films has been evaluated. Films were prepared by casting from solution and exposed to solutions of the crosslinkers in acetone-water solution. Swelling in water and in phosphate buffered saline (PBS) was then used to assess the effectiveness of the crosslinkers. GTA-crosslinked films were found to be of low stability compared with those treated with EDC, EX 810, and DVS. Results suggest that instability in GTA-crosslinked materials arises in part from residual acid catalyst. The effects of polymer molecular weight are not uniform. With GTA-crosslinked film produced from higher molecular weight HA swells more, and this is attributed to reduced diffusion of the crosslinker, but with EDC, the opposite effect is observed, implying some additional molecular weight dependent mechanism. Differential scanning calorimetry and dynamic mechanical thermal analysis results suggest that there are no significant structural difference between the gels for each crosslinker system and only the crosslink density and moisture content alters the transitions. (c) 2007 Wiley Periodicals, Inc.