AbstractsBiology & Animal Science

A chitosan/dextran-based (CD) hydrogel has been developed as a post-surgical aid for endoscopic sinus surgery, and has been successful in preventing adhesion formation in human and animal trials. Interest in extending the use of this hydrogel for abdominal and spinal surgeries has prompted the necessity to formulate a gel with reduced cytotoxicity. The CD hydrogel is formed by cross-linking between aldehyde groups present in dextran aldehyde (DA) and the amine groups on succinylated chitosan. The active component, DA, was formulated with a lower degree of oxidation (DA25) in anticipation of reduced cytotoxicity in order to comply with FDA standards prior to approval for use in vivo. DA100 (80% oxidised), and DA25 (25% oxidised) and the corresponding CD hydrogels (CD100 and CD25) were studied in four distinct phases. The first phase examined the antimicrobial activities of DA100, DA25, CD100 and CD25 in vitro against bacteria commonly causing post-operative abdominal infections. Broth micro-dilution test and time course growth experiments were performed. The reduction in oxidation of DA (from 80% to 25%) resulted in a decrease in antimicrobial efficacy in both the DA alone and CD hydrogel. Both formulations of DA were more effective against anaerobes than aerobic species. The second phase examined the mechanism of action of DA100 and CD100 against the anaerobic organism Bacteroides fragilis. Transmission electron microscopy revealed changes to the cell wall, i.e. cell wall rippling and disruption of cell septation following incubation of DA100 and CD100 at concentrations of 4 mg/mL and 20 mg/mL respectively. The third phase consisted of investigations into the chemical changes that occurred in the CD hydrogel over time. These studies were performed in order to determine the number of free aldehyde groups present. Gel proton nuclear magnetic resonance was performed and indicated a clear decrease in both the amine and aldehyde peaks as the gel components created cross-links; thereby, reducing the number of free aldehydes available for antimicrobial activity. Additionally resazurin reduction revealed that DA100 was more chemically active, possessing a greater number of free aldehyde groups than DA25, as indicated by a rapid redox reaction with concentrations of DA100 as low as 10 mg/mL; whereas concentrations up to 70 mg/mL of DA25 did not result in any colour changes. Lastly, biocompatibility assessments were also performed in order to compare and determine the suitability of the new formulation of DA (DA25) for future use in vivo. The following aspects were taken into account: the level of cytotoxicity in vitro of CD100 and CD25 when co-incubated with human dermal fibroblasts; the effect on cell migration and proliferation in in vitro wound healing assays; and the uptake of the CD hydrogel into dermal fibroblasts over time. To reduce possible issues with regulatory approval regarding cytotoxicity for use in vivo, surgical concentrations of the CD hydrogel for endoscopic sinus surgery (40 mg/mL (5% SC + 3% DA))…