Kinetics and isotherm of fibronectin adsorptionto three-dimensional porous chitosan scaffoldsexplored by 125I-radiolabelling

摘要

In this study, 125I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porouspolymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation inserum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and15%) were incubated in FN solutions with concentrations ranging from 5 to 50 μg/mL. The kinetic and isotherm of FNadsorption to CH were successfully followed using 125I-FN as a tracer molecule. While on DA 4% the levels of adsorbedFN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbedamounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for thesame FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%.Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FNby new FN molecules from serum than DA 15%. In accordance, when assessing the effcacy of passively adsorbed FN topromote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA15%, 5 mu g/mL of FN being already effcient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%.Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorptionto porous polymeric scaffolds, both from single and complex protein solutions.