Cellular responses to chitosan in vitro: the importance of deacetylation

Chitin and chitosan (a deacetylated derivative of chitin) have been proposed for biomedical applications because of their biocompatibility and abundance in nature. We have investigated the effect of the percentage of deacetylation (%DD) of chitosan on biocompatibility from two sources, shrimp and cuttle fish, with two cell lines, L929 and BHK21(C13). The difference in %DD for each source was approximately 10% in the range of 76-90%. Biocompatibility was investigated for: (1) cell adherence and growth on the chitosan samples as substrate; (2) the effect of extract media on 2d and 7d growth; and (3) the presence of an inhibition zone. The results were similar for both cell lines. The chitosan samples were air-dried on to tissue culture-grade petri dishes to provide a substrate for the adherent-cell cultures. The higher %DD substrates from each source supported attachment of the cells, while the lower %DD did not. Cells cultured in medium conditioned by each substrate (i.e. extract medium) displayed an initial difference in growth which was abrogated in cultures incubated for 7 days. No inhibition zone was apparent. However, after 7 days, some cells were noted migrating on to the low %DD substrate disks. The morphology of these cells was changed with the presence of pseudopodia being apparent. Thus, especially with regard to attachment the %DD has a very important effect on the biocompatibility of the chitosan and should be monitored carefully.