Attachment, spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation

Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs <= 13%. Regarding cell proliferation, cells grown on films with the lowest DA (4%) revealed a higher specific growth rate as compared to those grown on films with higher DAs. Films with a DA of 49% failed to sustain cell proliferation. In addition, a longer lag-phase was observed on Ch, as compared to TCPS, in accordance to an observed delay of cell spreading. The present findings revealed that differences in the DA as small as 9% may be critical in terms of the osteoblast response to two-dimensional Ch-based matrices.