Influence of freezing temperature and deacetylation degree on the performance of freeze-dried chitosan scaffolds towards cartilage tissue engineering

Chitosan-based porous structures have been significantly studied across the world as potential tissue engineering scaffolds. Despite the differences in chitosan produced from squid pens or crustacean shells, with the former being more reactive and easily available with a higher degree of deacetylation (DD), most of the studies report the use of crab or shrimp chitosan as they are readily available commercial sources. The aim of this work was to highlight the great potential of chitosan produced from squid pens for biomedical application. From freeze-dried scaffolds for soft tissue engineering, we investigated the influence of the DD of chitosan and the freezing temperature during processing on their performance. Chitosan was obtained by deacetylation of -chitin previously isolated from endoskeleton of giant squid Dosidicus gigas (DD 91.2%) and compared with a commercially available batch obtained from crab shells (DD 76.6%). Chitosan solutions were frozen at -80C or -196C and further freeze-dried to obtain 3D porous structures (scaffolds). Scaffolds prepared at -196C have a compact structure with smaller pores, while those prepared at -80C showed a lamellar structure with larger pores. The compressive modulus varied from 0.7 up to 8.8 MPa. Both types of scaffolds were stable on PBS, including in the presence of lysozyme, up to 4 weeks. Furthermore, the squid chitosan scaffolds processed at -80C promoted ATDC5 chondrocyte-like cells adhesion and proliferation. The results suggest that the developed squid chitosan scaffolds might be further exploited for applications in cartilage tissue engineering.